Mutated anthrax toxin protective antigen proteins that specifically target cells containing high amounts of cell-surface metalloproteinases or plasminogen activator receptors

ABSTRACT

The present invention provides methods of specifically targeting compounds to cells overexpressing matrix metalloproteinases, plasminogen activators, or plasminogen activator receptors, by administering a compound and a mutant protective antigen protein comprising a matrix metalloproteinase or a plasminogen activator-recognized cleavage site in place of the native protective antigen furin-recognized cleavage site, wherein the mutant protective antigen is cleaved by a matrix metalloproteinase or a plasminogen activator overexpressed by the cell, thereby translocating into the cell a compound comprising a lethal factor polypeptide comprising a protective antigen binding site.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is related to U.S. Pat. No. 5,591,631; U.S. Pat. No.5,677,274; and U.S. patent application Ser. No. 08/937,276, filed Sep.15, 1997, now U.S. Pat. No. 6,592,872; each herein incorporated byreference in its entirety. This application is a divisional applicationof U.S. patent application Ser. No. 12/288,482, filed Oct. 20, 2008, nowU.S. Pat. No. 8,791,074, which is a divisional application of U.S.patent application Ser. No. 10/088,952, filed on Mar. 22, 2002, now U.S.Pat. No. 7,468,352, which is the 371 national stage of PCT/US00/26192,filed Sep. 22, 2000, and claims the benefit of U.S. ProvisionalApplication No. 60/155,961, filed Sep. 24, 1999, the contents of whichis herein incorporated by reference in its entirety.

This application includes a Sequence Listing as a text file named“77867-911316-SEQLIST.txt” created Jun. 23, 2014, and containing 9453bytes. The material contained in this text file is incorporated byreference in its entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

Not applicable.

BACKGROUND OF THE INVENTION

Anthrax toxin is a three-part toxin secreted by Bacillus anthracisconsisting of protective antigen (PA, 83 kDa), lethal factor (LF, 90kDa) and edema factor (EF, 89 kDa) (Smith, H., et al., J. Gen.Microbiol., 29:517-521 (1962); Leppla, S. H., Sourcebook of bacterialprotein toxins, p. 277-302 (1991); Leppla, S. H., Handb. Nat. Toxins,8:543-572 (1995)), which are individually non-toxic. The mechanism bywhich individual toxin components interact to cause toxicity wasrecently reviewed (Leppla, S. H., Handb. Nat. Toxins, 8:543-572 (1995)).Protective antigen, recognized as central, receptor-binding component,binds to an unidentified receptor (Escuyer, V., et al., Infect. Immun.,59:3381-3386 (1991)) and is cleaved at the sequence RKKR₁₆₇ (SEQ IDNO:1) by cell-surface furin or furin-like proteases (Klimpel, K. R., etal., Proc. Natl. Acad. Sci. USA, 89:10277-10281 (1992); Molloy, S. S.,et al., J. B. Chem., 267:16396-16402 (1992)) into two fragments: PA63, a63 kDa C-terminal fragment, which remains receptor-bound; and PA20, a 20kDa N-terminal fragment, which is released into the medium (Klimpel, K.R., et al., Mol. Microbiol., 13:1094-1100 (1994)). Dissociation of PA20allows PA63 to form heptamer (Milne, J. C., et al., J. Biol. Chem.,269:20607-20612 (1994); Benson, E. L., et al., Biochemistry,37:3941-3948 (1998)) and also bind LF or EF (Leppla, S. H., et al.,Bacterial protein toxins, p. 111-112 (1988)). The resultinghetero-oligomeric complex is internalized by endocytosis (Gordon, V. M.,et al., Infect. Immun., 56:1066-1069 (1988)), and acidification of thevesicle causes insertion of the PA63 heptamer into the endosomalmembrane to produce a channel through which LF or EF translocate to thecytosol (Friedlander, A. M., J. Biol. Chem., 261:7123-7126 (1986)),where LF and EF induce cytotoxic events.

Thus, the combination of PA+LF, named anthrax lethal toxin, killsanimals (Beal, F. A., et al., J. Bacteriol., 83:1274-1280 (1962);Ezzell, J. W., et al., Infect. Immun., 45:761-767 (1984)) and certaincultured cells (Friedlander, A. M., J. Biol. Chem., 261:7123-7126(1986); Hanna, P. C., et al., Mol. Biol. Cell., 3:1267-1277 (1992)), dueto intracellular delivery and action of LF, recently proven to be azinc-dependent metalloprotease that is known to cleave at least twotargets, mitogen-activated protein kinase kinase 1 and 2 (Duesbery, N.S., et al., Science, 280:734-737 (1998); Vitale, G., et al., Biochem.Biophys. Res. Commun., 248:706-711 (1998)). The combination of PA+EF,named edema toxin, disables phagocytes and probably other cells, due tothe intracellular adenylate cyclase activity of EF (Leppla, S. H., Proc.Natl. Acad. Sci. USA., 79:3162-3166 (1982)).

LF and EF have substantial sequence homology in amino acid (aa) 1-250(Leppla, S. H., Handb. Nat. Toxins, 8:543-572 (1995)), and a mutagenesisstudy showed this region constitutes the PA-binding domain (Quinn, C.P., et al., J. Biol. Chem., 166:20124-20130 (1991)). Systematic deletionof LF fusion proteins containing the catalytic domain of Pseudomonasexotoxin A established that LF aa 1-254 (LFn) are sufficient to achievetranslocation of “passenger” polypeptides to the cytosol of cells in aPA-dependent process (Arora, N., et al., J. Biol. Chem., 267:15542-15548(1992); Arora, N., et al., J. Biol. Chem., 268:3334-3341 (1993)). Ahighly cytotoxic LFn fusion to the ADP-ribosylation domain ofPseudomonas exotoxin A, named FP59, has been developed (Arora, N., etal., J. Biol. Chem., 268:3334-3341 (1993)). When combined with PA, FP59kills any cell type which contains receptors for PA by the mechanism ofinhibition of initial protein synthesis through ADP ribosylatinginactivation of elongation factor 2 (EF-2), whereas native LF is highlyspecific for macrophages (Leppla, S. H., Handb. Nat. Toxins, 8:543-572(1995)). For this reason, FP59 is an example of a potent therapeuticagent when specifically delivered to the target cells with atarget-specific PA.

The crystal structure of PA at 2.1 A was solved by X-ray diffraction(PDB accession 1ACC) (Petosa, C., et al., Nature, 385:833-838 (1997)).PA is a tall, flat molecule having four distinct domains that can beassociated with functions previously defined by biochemical analysis.Domain 1 (aa 1-258) contains two tightly bound calcium ions, and a largeflexible loop (aa 162-175) that includes the sequence RKKR₁₆₇ (SEQ IDNO:1), which is cleaved by furin during proteolytic activation. Domain 2(aa 259-487) contains several very long β-strands and forms the core ofthe membrane-inserted channel. It is also has a large flexible loop (aa303-319) implicated in membrane insertion. Domain 3 (aa 488-595) has noknown function. Domain 4 (aa 596-735) is loosely associated with theother domains and is involved in receptor binding. For cleavage atRKKR₁₆₇ (SEQ ID NO:1) is absolutely required for the subsequent steps intoxin action, it would be of great interest to engineer it to thecleavage sequences of some disease-associated proteases, such as matrixmetalloproteinases (MMPs) and proteases of the plasminogen activationsystem (e.g., t-PA, u-PA, etc., see, e.g., Romer et al., APMIS107:120-127 (1999)), which are typically overexpressed in tumors.

MMPs and plasminogen activators are families of enzymes that play aleading role in both the normal turnover and pathological destruction ofthe extracellular matrix, including tissue remodeling (Birkedal-Hansen,H., Curr Opin Cell Biol, 7:728-735 (1995); Alexander, C. M., et al.,Development, 122:1723-1736 (1996)), angiogenesis (Schnaper, H. W, etal., J Cell Physiol, 156:235-246 (1993); Brooks, P. C., et al., Cell,92:391-400 (1998)), tumor invasion and metastasis formation. The membersof the MMP family are multidomain, zinc-containing, neutralendopeptidases and include the collagenases, stromelysins, gelatinases,and membrane-type metalloproteinases (Birkedal-Hansen, H., Curr OpinCell Biol, 7:728-735 (1995)). It has been well documented in recentyears that MMPs and proteins of the plasminogen activation system, e.g.,plasmiogen activator receptors and plasminogen activators, areoverexpressed in a variety of tumor tissues and tumor cell lines and arehighly correlated to the tumor invasion and metastasis (Crawford, H. C.,et al., Invasion Metastasis, 14:234-245 (1995); Garbisa, S., et al.,Cancer Res., 47:1523-1528 (1987); Himelstein, B. P., et al., Invest.Methods, 14:246-258 (1995); Juarez, J., et al., Int. J. Cancer, 55:10-18(1993); Kohn, E. C., et al., Cancer Res., 55:1856-1862 (1995); Levy, A.T., et al., Cancer Res., 51:439-444 (1991); Mignatti, P., et al.,Physiol. Rev., 73:161-195 (1993); Montgomery, A. M., et al., CancerRes., 53:693-700 (1993); Stetler-Stevenson, W. G., et al., Annu Rev CellBiol, 9:541-573 (1993); Stetler-Stevenson, W. G., Invest. Methods,14:4664-4671 (1995); Davidson, B., et al., Gynecol. Oncol., 73:372-382(1999); Webber, M. M., et al., Carcinogenesis, 20:1185-1192 (1999);Johansson, N., et al., Am J Pathol, 154:469-480 (1999); Ries, C., etal., Clin Cancer Res., 5:1115-1124 (1999); Zeng, Z. S., et al.,Carcinogenesis, 20:749-755 (1999); Gokaslan, Z. L., et al., Clin ExpMetastasis, 16:721-728 (1998); Forsyth, P. A., et al., Br J Cancer,79:1828-1835 (1999); Ozdemir, E., et al., J Urol, 161:1359-1363 (1999);Nomura, H., et al., Cancer. Res., 55:3263-3266 (1995); Okada, Y., etal., Proc. Natl. Acad. Sci. USA., 92:2730-2734 (1995); Sato, H., et al.,Nature, 370:61-65 (1994); Chen, W. T., et al., Ann NY Acad Sci,878:361-371 (1999); Sato, T., et al., Br J Cancer, 80:1137-43 (1999);Polette, M., et al., Int J Biochem cell Biol., 30:1195-1202 (1998);Kitagawa, Y., et al., J Urol., 160:1540-1545; Nakada, M., et al., Am JPathol., 154:417-428 (1999); Sato, H., et al., Thromb Haemost,78:497-500 (1997)).

Among the MMPs, MMP-2 (gelatinase A), MMP-9 (gelatinase B) andmembrane-type 1 MMP (MT1-MMP) are reported to be most related toinvasion and metastasis in various human cancers (Crawford, H. C., etal., Invasion Metastasis, 14:234-245 (1995); Garbisa, S., et al., CancerRes., 47:1523-1528 (1987); Himelstein, B. P., et al., Invest. Methods,14:246-258 (1995); Juarez, J., et al., Int. J. Cancer, 55:10-18 (1993);Kohn, E. C., et al., Cancer Res., 55:1856-1862 (1995); Levy, A. T., etal., Cancer Res., 51:439-444 (1991); Mignatti, P., et al., Physiol.Rev., 73:161-195 (1993); Montgomery, A. M., et al., Cancer Res.,53:693-700 (1993); Stetler-Stevenson, W. G., et al., Annu Rev Cell Biol,9:541-573 (1993); Stetler-Stevenson, W. G., Invest. Methods,14:4664-4671 (1995); Davidson, B., et al., Gynecol. Oncol., 73:372-382(1999); Webber, M. M., et al., Carcinogenesis, 20:1185-1192 (1999);Johansson, N., et al., Am J Pathol, 154:469-480 (1999); Ries, C., etal., Clin Cancer Res., 5:1115-1124 (1999); Zeng, Z. S., et al.,Carcinogenesis, 20:749-755 (1999); Gokaslan, Z. L., et al., Clin ExpMetastasis, 16:721-728 (1998); Forsyth, P. A., et al., Br J Cancer,79:1828-1835 (1999); Ozdemir, E., et al., J Urol, 161:1359-1363 (1999);Nomura, H., et al., Cancer. Res., 55:3263-3266 (1995); Okada, Y., etal., Proc. Natl. Acad. Sci. USA., 92:2730-2734 (1995); Sato, H., et al.,Nature, 370:61-65 (1994); Chen, W. T., et al., Ann NY Acad Sci,878:361-371 (1999); Sato, T., et al., Br J Cancer, 80:1137-43 (1999);Polette, M., et al., Int J Biochem cell Biol., 30:1195-1202 (1998);Kitagawa, Y., et al., J Urol., 160:1540-1545; Nakada, M., et al., Am JPathol., 154:417-428 (1999); Sato, H., et al., Thromb Haemost,78:497-500 (1997)). The important role of MMPs during tumor invasion andmetastasis is to break down tissue extracellular matrix and dissolutionof epithelial and endothelial basement membranes, enabling tumor cellsto invade through stroma and blood vessel walls at primary and secondarysites. MMPs also participate in tumor neoangiogenesis and areselectively upregulated in proliferating endothelial cells in tumortissues (Schnaper, H. W, et al., J Cell Physiol, 156:235-246 (1993);Brooks, P. C., et al., Cell, 92:391-400 (1998); Chambers, A. F., et al.,J Natl Cancer Inst, 89:1260-1270 (1997)). Furthermore, these proteasescan contribute to the sustained growth of established tumor foci by theectodomain cleavage of membrane-bound pro-forms of growth factors,releasing peptides that are mitogens for tumor cells and/or tumorvascular endothelial cells (Arribas, J., et al., J Biol Chem,271:11376-11382 (1996); Suzuki, M., et al., J Biol Chem, 272:31730-31737(1997)).

However, catalytic manifestations of MMP and plasminogen activators arehighly regulated. For example, the MMPs are expressed as inactivezymogen forms and require activation before they can exert theirproteolytic activities. The activation of MMP zymogens involvessequential proteolysis of N-terminal propeptide blocking the active sitecleft, mediated by proteolytic mechanisms, often leading to anautoproteolytic event (Springman, E. B., et al., Proc Natl Acad Sci USA,87:364-368 (1990); Murphy, G., et al., APMIS, 107:38-44 (1999)). Second,a family of proteins, the tissue inhibitors of metalloproteinases(TIMPs), are correspondingly widespread in tissue distribution andfunction as highly effective MMP inhibitors (Ki˜10⁻¹⁰ M)(Birkedal-Hansen, H., et al., Crit Rev Oral Biol Med, 4:197-250 (1993)).Though the activities of MMPs are tightly controlled, invading tumorcells that utilize the MMP's degradative capacity somehow circumventthese negative regulatory controls, but the mechanisms are not wellunderstood.

The contributions of MMPs in tumor development and metastatic processlead to the development of novel therapies using synthetic inhibitors ofMMPs (Brown, P. D., Adv Enzyme Regul, 35:293-301 (1995);Wojtowicz-Praga, S., et al., J Clin Oncol, 16:2150-2156 (1998);Drummond, A. H., et al., Ann NY Acad Sci, 30:228-235 (1999)). Among amultitude of synthetic inhibitors generated, Marimastat is alreadyclinically employed in cancer treatment (Drummond, A. H., et al., Ann NYAcad Sci, 30:228-235 (1999)).

Here, as an alternate to the use of MMP inhibitors, we explored a novelstrategy using modified PAs which could only be activated by MMPs orplasminogen activators to specially kill MMP- or and plasminogenactivators-expressing tumor cells. PA mutants are constructed in whichthe furin recognition site is replaced by sequences susceptible tocleavage by MMPs or and plasminogen activators. When combined with LF oran LF fusion protein comprising the PA binding site, these PA mutantsare specifically cleaved by cancer cells, exposing the LF binding siteand translocating the LF or LF fusion protein into the cell, therebyspecifically delivering a compounds, e.g., a therapeutic or diagnosticagent, to the cell.

SUMMARY OF THE INVENTION

Matrix metalloproteinases (“MMPs”) and proteins of the plasminogenactivation system (e.g., t-PAR, u-PAR, u-PA, t-PA) are overexpressed ina variety of tumor tissues and tumor cell lines and are highlycorrelated to tumor invasion and metastasis. In addition, these proteinsare overexpressed in other cells such as inflammatory cells. Here weconstructed anthrax toxin protective antigen (PA) mutants, in which thefurin site is replaced by sequences specifically cleaved by MMPs orplasminogen activators. These MMP or plasminogen activator targeted PAmutants are only activated by plasminogen activator- or MMP-expressingtumor cells, so as to specifically deliver a toxin, a diagnostic, or atherapeutic agent. The activation occurs primarily on the cell surface,resulting in translocation and delivery of the compounds. The compoundscan be diagnostic or therapeutic agents. Preferably the compounds aredelivered to the cells of a human subject suffering from cancer, therebykilling the cancer cells and treating the cancer.

In one aspect, the present invention provides a method of targeting acompound to a cell over-expressing a matrix metalloproteinase, aplasminogen activator, or a plasminogen activator receptor, the methodcomprising the steps of: (i) administering to the cell a mutant PAprotein comprising a matrix metalloproteinase or a plasminogenactivator-recognized cleavage site in place of the native PAfurin-recognized cleavage site, wherein the mutant PA is cleaved by amatrix metalloproteinase or a plasminogen activator; and (ii)administering to the cell a compound comprising an LF polypeptidecomprising a PA binding site; wherein the LF polypeptide binds tocleaved PA and is translocated into the cell, thereby delivering thecompound to the cell.

In one embodiment, the cell overexpresses a matrix metalloproteinase. Inanother embodiment, the matrix metalloproteinase is selected from thegroup consisting of MMP-2 (gelatinase A), MMP-9 (gelatinase B) andmembrane-type 1 MMP (MT1-MMP). In another embodiment, the matrixmetalloproteinase-recognized cleavage site is selected from the groupconsisting of GPLGMLSQ (SEQ ID NO:2) and GPLGLWAQ (SEQ ID NO:3).

In one embodiment, the cell overexpresses a plasminogen activator or aplasminogen activator receptor. In another embodiment, the plasminogenactivator is selected from the group consisting of t-PA (tissue-typeplasminogen activator) and u-PA (urokinase-type plasminogen activator).In another embodiment, the plasminogen activator-recognized cleavagesite is selected from the group consisting of PCPGRVVGG, PGSGRSA,PGSGKSA, and PQRGRSA (SEQ ID NOS:4-7, respectively).

In one embodiment, the cell is a cancer cell. In another embodiment, thecancer is selected from the group consisting of lung cancer, breastcancer, bladder cancer, thyroid cancer, liver cancer, lung cancer,pleural cancer, pancreatic cancer, ovarian cancer, cervical cancer,colon cancer, fibrosarcoma, neuroblastoma, glioma, melanoma, monocyticleukemia, and myelogenous leukemia. In another embodiment, the cell isan inflammatory cell. In another embodiment, the cell is a human cell.

In one embodiment, the lethal factor polypeptide is native lethalfactor. In another embodiment, the compound is native lethal factor.

In one embodiment, the lethal factor polypeptide is linked to aheterologous compound. In another embodiment, the compound is adiagnostic or a therapeutic agent. In another embodiment, the compoundis shiga toxin, A chain of diphtheria toxin, or Pseudomonas exotoxin A.In another embodiment, the compound is a detectable moiety or a nucleicacid.

In one embodiment, the compound is covalently linked to lethal factorvia a chemical bond. In another embodiment, the heterologous compound isrecombinantly linked to lethal factor.

In one embodiment, the mutant PA protein is a fusion protein comprisinga heterologous receptor binding domain. In another embodiment, theheterologous receptor binding domain is selected from the groupconsisting of a single chain antibody and a growth factor.

In one aspect, the present invention provides an isolated mutantprotective antigen protein comprising a matrix metalloproteinase or aplasminogen activator-recognized cleavage site in place of the nativeprotective antigen furin-recognized cleavage site, wherein the mutantprotective antigen is cleaved by a matrix metalloproteinase or aplasminogen activator.

In one embodiment, the matrix metalloproteinase or a plasminogenactivator-recognized cleavage site is selected from the group consistingof PCPGRVVGG, PGSGRSA, PGSGKSA, PQRGRSA, GPLGMLSQ and GPLGLWAQ (SEQ IDNOS:4-7, 2 and 3, respectively).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Generation of PA mutants can be specifically processed by MMPs.(A). Schematic representation of MMP substrate PA mutants. The furincleavage site RKKR (SEQ ID NO:1) was replaced with gelatinase favoritesubstrate sequences GPLGMLSQ (SEQ ID NO:2) in PA-L1 and GPLGLWAQ (SEQ IDNO:3) in PA-L2. The arrows show the cleavage sites of furin or MMPs asindicated. (B). Cleavage of PA-L1 by MMP-2, MMP-9 and soluble formfurin. As described in Materials and Methods, PA-L1 was incubated withMMP-2, MMP-9 and furin, respectively, aliquots were withdrawn at thetime points indicated, and the samples were analyzed by western blottingwith the rabbit polyclonal antibody against PA. (C). Cleavage of PA-L2by MMP-2, MMP-9 and soluble form furin. PA-L2 was treated as in B. (D).Cleavage of WT-PA by MMP-2, MMP-9 and soluble form furin. WT-PA wastreated as in B.

FIG. 2. Zymographic analysis of the gelatinases associated withserum-free conditioned media (A) or Triton X-100 extracts (B) of Verocells, HT1080 cells and A2058 cells. 1 mg of cell extract protein, orvolumes of conditioned medium (3-4 ml) normalized to the proteinconcentration of the corresponding cell extracts were analyzed bygelatin zymography as described in Materials and Methods.

FIG. 3. Cytotoxicity of PA-L1 and PA-L2 (A) or nicked form of them (B)to the MMP non-expressing Vero cells. As described in Materials andMethods, Vero cells were cultured in 96-well plates to 80-100%confluence washed and replaced with serum-free DMEM medium. Thendifferent concentrations (from 0 to 1000 ng/ml) of WT-PA, PA-L1 andPA-L2, or MMP-2 nicked PA-L1 and PA-L2 combined with FP59 (constant at50 ng/ml) were separately added to the cells. The toxins were left inthe medium for 48 hours, or removed and replaced with freshserum-containing DMEM after 6 hour. MTT was added to determined cellviability at 48 hours. Nicked PA-L1 and PA-L2 were prepared by cleavageof PA-L1 and PA-L2 by active MMP-2 at 37° C. for 3 hours as described inMaterials and Methods.

FIG. 4. Cytotoxicity of PA-L1 and PA-L2 to the MMP expressing tumorHT1080 cells (A), A2058 cells (B) and MDA-MB-231 cells (C). As describedin Materials and Methods, HT1080 and A2058 cells were cultured to80-100% confluence, washed and replaced with serum-free DMEM medium.Then different concentrations (from 0 to 1000 ng/ml) of WT-PA, PA-L1 andPA-L2 combined with FP59 (constant at 50 ng/ml) were separately added tothe cells and incubated for 6 hours and 48 hours. MTT was added todetermined cell viability at 48 hours.

FIG. 5. Effect of MMP inhibitors on cytotoxicity of PA-L1 and PA-L2 toHT1080 cells. HT1080 cells were cultured to 80% confluence in a 96-wellplate, and washed twice with serum-free DMEM. Then MMP inhibitorsGM6001, BB94 and BB2516 were added to the cells at final concentrationof 10 μM in serum-free DMEM. After 300 min pre-incubation with the MMPinhibitors, WT-PA, PA-L1 and PA-L2 (300 ng/ml) combined with FP59 (50ng/ml) were separately added to the cells and incubated for 6 hours.After that, the medium containing the toxins and MMP inhibitors wereremoved, and fresh serum-containing medium was added and incubationcontinued to 48 hours. MTT was added to determine cell viability asdescribed in Materials and Methods.

FIG. 6. PA-L1 and PA-L2 selectively killed MMP-expressing tumor cells ina co-culture model. As described in Materials and Methods, Vero, HT1080,MDA-MB-231 and A2058 cells were cultured in the separate chambers of8-chamber slides to 80 to 100% confluence. Then the slides withpartitions removed were put into 100 mm petri dishes with serum-freemedium, so that the different cells were in the same cultureenvironment. WT-PA, PA-L1 or PA-L2 (300 ng/ml) each combined with FP59(50 ng/ml) were separately added to the cells, and incubated to 48hours. MTT was added to determine cell viability. Insert, after 48 hourstoxin challenge MTT was added to the cells, live cells converted MTT toblue dye, which precipitated in cytosol, while dead cells remainedcolorless.

FIG. 7. Binding and activation processing of PA, PA-L1 and PA-L2 on thecell surface of Vero (A) and HT1080 (B) cells. As described in Materialsand Methods, Vero and HT1080 cells were cultured in 24-well plates to80-100% of confluence, washed and changed serum-free media. Then PA,PA-L1 and PA-L2 were added to the cells with a final concentration of1000 ng/ml, incubated for different times (0, 10 min, 40 min, 120 minand 360 min). The cell lysates were prepared for western blottinganalysis using rabbit anti-PA polyclonal antibody (#5308) to check theprocessing status of PA and PA mutants.

FIG. 8. The role of transfected MT1-MMP in cytotoxicity of PA-L1 andPA-L2 to COS-7 cells. A. Cytotoxicity of PA-L1 and PA-L2 to COS-7 cells.As described in Materials and Methods, COS-7 cells were cultured to80-100% of confluence, washed and replaced with serum-free DMEM medium.Then different concentrations (from 0 to 1000 ng/ml) of WT-PA, PA-L1 andPA-L2 combined with FP59 (constant at 50 ng/ml) were separately added tothe cells and incubated for 6 hours and 48 hours. MTT was added todetermined cell viability at 48 hours. Insert: Zymographic analysis ofcell extracts and culture supernatants of COS-7 as described inMaterials and Methods, using supernatant of HT1080 as control. B.Cytotoxicity of PA-L1 and PA-L2 to CosgMT1. CosgMT1 cells were treatedthe same as in A. Insert: Comparison expression of MT1-MMP from COS-7and CosgMT1 cells by western blotting using a rabbit anti-MT1-MMPantibody (AB815, CHEMICON International, Inc.).

FIG. 9. Generation of mutated PA proteins which can be specificallycleaved by uPA or tPA. Cleavage of PA and mutated PA proteins by solubleform of furin (in panel a), uPA (in panel b) or tPA (in panel c).Proteins were incubated with furin, uPA or tPA, for the times indicatedand samples were analyzed by SDS-PAGE and Commassie staining in panel a,or diluted and analyzed by Western blotting with rabbit polyclonalantibody against PA in panel b and c.

FIG. 10. Binding and processing of pro-uPA by different cell lines. Verocells, Hela cells, A2058 cells, and Bowes cells were cultured in 24-wellplate to confluence, washed and incubated in serum-free media with 1μg/ml of pro-uPA and 1 μg/ml of glu-plasminogen for 1 h, then the celllysates were prepared for Western blotting analysis with monoclonalantibody against uPA B-cahin (#394).

FIG. 11. Cytotoxicity of mutated PA proteins for uPAR expressing tumorcells. Hela cells (in panel a), A2058 cells (in panel b), and Bowescells (in panel c) were cultured to 50% confluence, washed and replacedwith serum-free DMEM containing 100 ng/ml of pro-uPA and 1 μg/ml ofglu-plasminogen. Then different concentrations (from 0 to 1000 ng/ml) ofPA, PA-U1, PA-U2, PA-U3, PA-U4, and PA-U7 together with FP59 (constantat 50 ng/ml) were incubated with the cells for 6 h. Then the toxins wereremoved and replaced with fresh serum-containing DMEM. MTT was added todetermined cell viability at 48 h.

FIG. 12. Cytotoxicity of mutated PA proteins for uPAR non-expressingVero cells. a. Vero cells were cultured in 96-well plates to 50%confluence, washed and replaced with serum-free DMEM containing 100ng/ml of pro-uPA and 1 μg/ml of glu-plasminogen. Then the cells weretreated with toxins as above. B. Vero cells were treated as in panel a,except that nicked PA-U2 was used for the cytotoxicity assay. NickedPA-U2 was prepared by cleavage of PA-U2 with uPA at 37° C. for 1 h asdescribed in Materials and Methods.

FIG. 13. Binding and proteolytic activation of PA and PA-U2 on thesurface of Vero cells (in panel a) and Hela (in panel b) cells. Vero andHela cells were cultured in 24-well plates to confluence, washed andchanged serum-free medium containing 100 ng/ml of pro-uPA and 1 μg/ml ofplasminogen with or without PM-1 (2 μg/ml). Then PA and PA-U2 were addedto the cells with a final concentration of 1000 ng/ml, incubated for 30min or 120 min. The cell lysates were prepared for Western blottinganalysis using rabbit anti-PA polyclonal antibody (#5308) to check theprocessing status of PA and PA-U2 and the effect of PAI-1 on it.

FIG. 14. Effects of PAI-1 on cytotoxicity of PA-U2 to tumor cells. Helacells (in panel a), A2058 cells (in panel b), and Bowes cells (in panelc) were cultured to 50% confluence in a 96-well plate, washed andincubated with serum-free DMEM containing 100 ng/ml of pro-uPA and 1μg/ml of glu-plasminogen with or without 2 μg/ml of PAI-1, for 30 min.Then different concentrations of PA and PA-U2 (from 0 to 1000 ng/ml)combined with FP59 (50 ng/ml) were separately added to the cells andincubated for 6 hours. After that, the toxins were removed and replacedwith fresh serum-containing DMEM. MTT was added to determined cellviability at 48 h.

FIG. 15. Effects of blocking uPAR on cytotoxicity of PA-U2 to the tumorcells. a. Effects of ATE on cytotoxicity of PA-U2 to Hela, A2058, andBowes cells. b. Effects of uPAR blocking antibody R3 on cytotoxicity ofPA-U2 to Hela, A2058, and Bowes cells. Cells were cultured to 50%confluence, washed and incubated with serum-free DMEM containing 100ng/ml of pro-uPA and 1 μg/ml of glu-plasminogen, and differentconcentrations of ATF or uPAR blocking antibody R3. Then PA and PA-U2(300 ng/ml each) combined with FP59 (50 ng/ml) were added to the cellsand incubated for 6 hours. After that, the toxins were removed andreplaced with fresh serum-containing DMEM. MTT was added to determinedcell viability at 48 h.

FIG. 16. PA-U2 selectively killed uPAR-expressing Hela cells in aco-culture model. Vero and Hela cells were cultured in the separatechambers of 8-chamber slides to confluence. Then the slides withpartitions removed were put into 100 mm petri dishes with serum-freemedium containing 100 ng/ml of pro-uPA and 1 μg/ml of glu-plasminogen,so that the different cells were in the same culture environment. PA andPA-U2 (1000 ng/ml) each combined with FP59 (50 ng/ml) were separatelyadded to the cells, and incubated to 48 hours. MTT was added todetermine cell viability. Insert, PA-U2 was selectively proteolyticallyactivated on Hela cells in a co-culture model. The cells were treatedthe same as in A, except that after 2 h incubation with toxins the cellswere washed and lysed, and the processing status of PA proteins weredetected by anti-PA antibody as in FIG. 14.

FIG. 17. Cytotoxicity of PA-U2, PA-U3, and PA-U4 on tPA expressingcells. Bowes cells (a) and HUVEC cells (b) were cultured to 50%confluence, washed and replaced with serum-free DMEM without pro-uPA andglu-plasminogen. Then the cells were treated with differentconcentrations (from 0 to 1000 ng/ml) of PA, PA-U2, PA-U3, and PA-U4together with FP59 (constant at 50 ng/ml) for 12 h. MTT was added todetermine cell viability at 48 h.

DETAILED DESCRIPTION

I. Introduction

Proteolytic degradation of the extracellular matrix plays a crucial roleboth in cancer invasion and non-neoplastic tissue remodeling, and inboth cases it is accomplished by a number of proteases. Best known arethe plasminogen activation system that leads to the formation of theserine protease plasmin, and a number of matrix metalloproteinase,including collagenases, gelatinases and stromelysins (Dano, K., et al.,APMIS, 107:120-127 (1999)). The close association between MMP andplasminogen activator overexpression and tumor metastasis has beennoticed for a decade. For example, the contributions of MMPs in tumordevelopment and metastatic process lead to the development of noveltherapies using synthetic inhibitors of MMPs (Brown, P. D., Adv EnzymeRegul, 35:293-301 (1995); Wojtowicz-Praga, S., et al., J Clin Oncol,16:2150-2156 (1998); Drummond, A. H., et al., Ann NY Acad Sci,30:228-235 (1999)). However, these inhibitors only slow growth and donot eradicate the tumors. The present study is the first effort to usebacterial toxins modified to target MMPs and plasminogen activators,which are highly expressed and employed by tumor cells for invasion.Mutant PA molecules in which the furin cleavage site is replaced by anMMP or plasminogen activator target site can be used to delivercompounds such as toxins to the cell, thereby killing the cell. Thecompounds have the ability to bind PA through their interaction with LFand are translocated by PA into the cell. The PA and LF-comprisingcompounds are administered to cells or subjects, preferably mammals,more preferably humans, using techniques known to those of skill in theart. Optionally, the PA and LF-comprising compounds are administeredwith a pharmaceutically acceptable carrier.

The compounds typically are either native LF or an LF fusion protein,i.e., those that have a PA binding site (approximately the first 250amino acids of LF, Arora et al., J. Biol. Chem. 268:3334-3341 (1993))fused to another polypeptide or compound so that the protein or fusionprotein binds to PA and is translocated into the cell, causing celldeath (e.g., recombinant toxin FP59, anthrax toxin lethal factor residue1-254 fusion to the ADP-ribosylation domain of Pseudomonas exotoxin A).The fusion is typically chemical or recombinant. The compounds fused toLF include, e.g., therapeutic or diagnostic agent, e.g., native LF, atoxin, a bacterial toxin, shiga toxin, A chain of diphtheria toxin,Pseudomonas exotoxin A, a protease, a growth factor, an enzyme, adetectable moiety, a chemical compound, a nucleic acid, or a fusionpolypeptide, etc.

The mutant PA molecules of the invention can be further targeted to aspecific cell by making mutant PA fusion proteins. In these mutantfusion proteins, the PA receptor binding domain is replaced by a proteinsuch as a growth factor or other cell receptor ligand specificallyexpressed on the cells of interest. In addition, the PA receptor bindingdomain may be replaced by an antibody that binds to an antigenspecifically expressed on the cells of interest.

These proteins provide a way to specifically kill tumor cells withoutserious damage to normal cells. This method can also be applied tonon-cancer inflammatory cells that contain high amounts of cell-surfaceassociated MMPs or plasminogen activators. These PA mutants are thususeful as therapeutic agents to specifically kill tumor cells.

We constructed two PA mutants, PA-L1 and PA-L2, in which the furinrecognition site is replaced by sequences susceptible to cleavage byMMPs, especially by MMP-2 and MMP-9. When combined with FP59, these twoPA mutant proteins specifically killed MMP-expressing tumor cells, suchas human fibrosarcoma HT1080 and human melanoma A2058, but did not killMMP non-expressing cells. Cytotoxicity assay in the co-culture model, inwhich all the cells were in the same culture environment and were equalaccessible to the toxins in the supernatant, showed PA-L1 and PA-L2specifically killed only MMP-expressing tumor cells HT1080 and A2058,not Vero cells. This result demonstrated activation processing of PA-L1and PA-L2 mainly occurred on the cell surfaces and mostly contributed bythe membrane-associated MMPs, so the cytotoxicity is restricted toMMP-expressing tumor cells. TIMPs are widely present in extracellularmilieu and inhibit MMP activity in supernatants. PA proteins bind to thecells very quickly with maximum binding happened within 60 min. Incontrast to secreted MMPs, membrane-associated MMPs express theirproteolytic activities more efficiently by anchoring on cell membraneand enjoying two distinct advantageous properties, which are highlyfocused on extracellular matrix substrates and more resistant toproteinase inhibitors present in extracellular milieu.

Recently it has been shown physiological concentrations of plasmin canactivate both MMP-2 and MMP-9 on cell surface of HT1080 by a mechanismindependent of MMP or acid proteinase activities (Mazzieri, R., et al.,EMBO J., 16:2319-2332 (1997)). In contrast, in soluble phase plasmindegrades both MMP-2 and MMP-9 (Mazzieri, R., et al., EMBO J.,16:2319-2332 (1997)). Thus, plasmin may provide a mechanism keepinggelatinase activities on cell surface to promote cell invasion. It hasbeen well established MT1-MMP functions as both activator and receptorof MMP-2, but has no effect on MMP-9 (see review Polette, M., et al.,Int J Biochem cell Biol., 30:1195-1202 (1998); Sato, H., et al., ThrombHaemost, 78:497-500 (1997)). A MMP-2/TIMP-2 complex binds to MT1-MMP oncell surface, which serves as a high-affinity site, then beproteolytically activated by an adjacent MT1-MMP, which serves as anactivator. Recent works have shown that adhesion receptors, such as αvβ3integrin (Brooks, P. C., et al., Cell, 85:683-693 (1996)) and cellsurface hyaluronan receptor CD44 (Tu, Q., et al, Gene Development,13:35-48 (1999)), may provide means to retain soluble active MMP-2 orMMP-9 to invasive tumor cell surface, where their proteolytic activitiesare most likely to promote cell invasion. For MMP activities involved intumor invasion and metastasis are localized and/or modulated on the cellsurface in insoluble phase, this makes MMPs an ideal target for tumortissues.

It was originally thought that the role of MMPs and plasminogenactivators was simply to break down tissue barriers to promote tumorinvasion and metastasis. It is now understood, for example, that MMPsalso participate in tumor neoangiogenesis and are selectivelyupregulated in proliferating endothelial cells. Therefore, thesemodified bacterial toxins may have the advantageous properties thattargeted to not only tumor cells themselves but may also the dividingvascular endothelial cells which essential to neoangiogenesis in tumortissues. Therefore, the MMP targeted toxins may also kill tumor cells bystarving the cells of necessary nutrients and oxygen.

The mutant PA molecules of the invention can also be specificallytargeted to cells using mutant PA fusion proteins. In these fusionproteins, the receptor binding domain of PA is replaced with aheterologous ligand or molecule such as an antibody that recognizes aspecific cell surface protein. PA protein has four structurally distinctdomains for performing the functions of receptor binding andtranslocation of the catalytic moieties across endosomal membranes(Petosa, C., et al., Nature, 385:833-838 (1997)). Domain 4 is thereceptor-binding domain and has limited contacts with other domains(Petosa, C., et al., Nature, 385:833-838 (1997)). Therefore, PA can bespecifically targeted to alternate receptors or antigens specificallyexpressed by tumors by replacing domain 4 with the targeting molecules,such as single-chain antibodies or a cytokines used by other immuntoxins(Thrush, G. R., et al., Annu Rev Immunol, 14:49-71 (1996)). For example,PA-L1 and PA-L2 are directed to alternate receptors, such as GM-CSFreceptor, which is highly expressed in leukemias cells and solid tumorsincluding renal, lung, breast and gastrointestinal carcinomas (Thrush,G. R., et al., Annu Rev Immunol, 14:49-71 (1996); 74-79). It should behighly expected that the combination of these two independent targetingmechanism should allow tumors to be more effectively targeted, and sideeffects such as hepatotoxicity and vascular leak syndrome should besignificantly reduced.

With respect to the plasminogen activation system, two plasminogenactivators are known, the urokinase-type plasminogen activator (uPA) andthe tissue-type plasminogen activator (tPA), of which uPA is the oneprimarily involved in extracellular matrix degradation (Dano, K., etal., APMIS, 107:120-127 (1999)). uPA is a 52 kDa serine protease whichis secreted as an inactive single chain proenzyme (pro-uPA) (Nielsen, L.S., et al., Biochemistry, 21:6410-6415 (1982); Petersen, L. C., et al.,J. Biol. Chem., 263:11189-11195 (1988)). The binding domain of pro-uPAis the epidermal growth factor-like amino-terminal fragment (ATF; aa1-135, 15 kDa) that binds with high affinity (Kd=0.5 mM) tourokinase-type plasminogen activator receptor (uPAR) (Cubellis, M. V.,et al., Proc. Natl. Acad. Sci. U.S.A., 86:4828-4832 (1989)), aGPI-linked receptor. uPAR is a 60 kDa three domain glycoprotein whoseN-terminal domain 1 contains the high affinity binding site for ATF ofpro-uPA (Ploug, M., et al., J. Biol. Chem., 266:1926-1933 (1991);Behrendt, N., et al., J. Biol. Chem., 266:7842-7847 (1991)). uPAR isoverexpressed on a variety of tumors, including monocytic andmyelogenous leukemias (Lanza, F., et al., Br. J. Haematol., 103:110-123(1998); Plesner, T., et al., Am. J. Clin. Pathol., 102:835-841 (1994)),and cancers of the breast (Carriero, M. V., et al., Clin. Cancer Res.,3:1299-1308 (1997)), bladder (Hudson, M. A., et al., J. Natl. CancerInst., 89:709-717 (1997)), thyroid (Ragno, P., et al., Cancer Res.,58:1315-1319 (1998)), liver (De Petro, G., et al., Cancer Res.,58:2234-2239 (1998)), pleura (Shetty, S., et al., Arch. Biochem.Biophys., 356:265-279 (1998)), lung (Morita, S., et al., Int. J. Cancer,78:286-292 (1998)), pancreas (Taniguchi, T., et al., Cancer Res.,58:4461-4467 (1998)), and ovaries (Sier, C. F., et al., Cancer Res.,58:1843-1849 (1998)). Pro-uPA binds to uPAR by ATF, while the bindingprocess does not block the catalytic, carboxyl-terminal domain. Byassociation with uPAR, pro-uPA gets near to and subsequently activatedby trace amounts of plasmin bound to the plasma membrane by cleavage ofthe single chain pro-uPA within an intra-molecular loop held closed by adisulfide bridge. Thus the active uPA consists of two chains (A+B) heldtogether by this disulfide bond (Ellis, V., et al., J. Biol. Chem.,264:2185-2188 (1989)).

Plasminogen is present at high concentration (1.5-2.0 μM) in plasma andinterstitial fluids (Dano, K., et al., Adv. Cancer Res., 44:139-266(1985)). Low affinity, high capacity binding of plasminogen tocell-surface proteins through the lysine binding sites of plasminogenkringles enhances considerably the rate of plasminogen activation by uPA(Ellis, V., et al., J. Biol. Chem., 264: 2185-2188 (1989); Stephens, R.W., et al., J. Cell Biol., 108:1987-1995 (1989)). Active uPA has highspecificity for Arg560-Val561 bond in plasminogen, and cleavage betweenthese residues gives rise to more plasmin that is referred to as“reciprocal zymogen activation” (Petersen, L. C., Eur. J. Biochem.,245:316-323 (1997)). The result of this system is efficient generationof active uPA and plasmin on cell surface. In this context, uPAR servesas a template for binding and localization of pro-uPA near to itssubstrate plasminogen on plasma membrane.

Unlike uPA, plasmin is a relatively non-specific protease, cleaving manyglycoproteins and proteoglycans of the extracellular matrix, as well asfibrin (Liotta, L. A., et al., Cancer Res., 41:4629-4636 (1981)).Therefore, cell surface bound plasmin mediates the non-specific matrixproteolysis which facilitates invasion and metastasis of tumor cellsthrough restraining tissue structures. In addition, plasmin can activatesome of the matrix metalloproteases which also degrade tissue matrix(Werb, Z., et al., N. Engl. J. Med., 296:1017-1023 (1977); DeClerck, Y.A., et al., Enzyme Protein, 49:72-84 (1996)). Plasmin can also activategrowth factors, such as TGF-β, which may further modulate stromalinteractions in the expression of enzymes and tumor neo-angiogenesis(Lyons, R. M., et al., J. Cell Biol., 106:1659-1665 (1988)). Plasminogenactivation by uPA is regulated by two physiological inhibitors,plasminogen activator inhibitor-1 and 2 (PAI-1 and PAI-2) (Cubellis, M.V., et al., Proc. Natl. Acad. Sci. U.S.A., 86:4828-4832 (1989); Ellis,V., et al., J. Biol. Chem., 265:9904-9908 (1990); Baker, M. S., et al.,Cancer Res., 50:4676-4684 (1990)), by formation 1:1 complex with uPA.Plasmin generated in the cell surface plasminogen activation system isrelatively protected from its principle physiological inhibitorα2-antiplasmin (Ellis, V., et al., J. Biol. Chem., 266:12752-12758(1991)).

Cancer invasion is essentially a tissue remodeling process in whichnormal tissue is substituted with cancer tissue. Accumulated data frompreclinical and clinical studies strongly suggested that the plasminogenactivation system plays a central role in the processes leading to tumorinvasion and metastasis (Andreasen, P. A., et al., Int. J. Cancer,72:1-22 (1997); Chapman, H. A., Curr. Opin. Cell Biol., 9:714-724(1997); Schmitt, M., et al., Thromb. Haemost., 78:285-296 (1997)). Highlevels of uPA, uPAR and PAL-1, but decreased PAI-2 are associated withpoor disease outcome (Schmitt, M., et al., Thromb. Haemost., 78:285-296(1997)). In situ hybridization studies of the tumor tissues have shownusually cancer cells highly expressed uPAR, while tumor stromal cellsexpressed pro-uPA, which subsequently binds to uPAR on the surface ofcancer cells where it is activated and thereby generating plasmin (Pyke,C., et al., Am. J. Pathol., 138:1059-1067 (1991)). For the activation ofpro-uPA is highly restricted to the tumor cell surface, it may be anideal target for cancer treatment.

uPA and tPA possess an extremely high degree of structure similarity(Lamba, D., et al., J. Mol. Biol., 258:117-135 (1996); Spraggon, G., etal., Structure, 3:681-691 (1995)), share the same primary physiologicalsubstrate (plasminogen) and inhibitors (PAI-1 and PAI-2) (Collen, D., etal., Blood, 78:3114-3124 (1991)), and exhibit restricted substratespecificity. By using substrate phage display and substrate subtractionphage display approaches, recent investigations had identifiedsubstrates that discriminate between uPA and tPA, showing the consensussubstrate sequences with high selectivity by uPA or tPA (Ke, S. H., etal., J. Biol. Chem., 272:20456-20462 (1997); Ke, S. H., et al., J. Biol.Chem., 272:16603-16609 (1997)). To exploit the unique characteristics ofthe uPA plasminogen system and anthrax toxin in the design of tumor cellselective cytotoxins, in the work described here, mutated anthrax PAproteins were constructed in which the furin site is replaced bysequences susceptible to specific cleavage by uPA. TheseuPAR/uPA-targeted PA proteins were activated selectively on the surfaceof uPAR-expressing tumor cells in the present of pro-uPA, and causedinternalization of a recombinant cytotoxin FP59 to selectively kill thetumor cells. Also, a mutated PA protein was generated which selectivelykilled tissue-type plasminogen activator expressing cells.

II. Methods of Producing PA and LF Constructs

A. Construction Nucleic Acids Encoding PA Mutants, LF, and PA and LFFusion Proteins

PA includes a cellular receptor binding domain, a translocation domain,and an LF binding domain. The PA polypeptides of the invention have atleast a translocation domain and an LF binding domain. In the presentinvention, mature PA (83 kDa) is one preferred embodiment. In additionto full length recombinant PA, amino-terminal deletions up to the 63 kDacleavage site or additions to the full length PA are useful. Arecombinant form of processed PA is also biologically active and couldbe used in the present invention. PA fusion proteins in which thereceptor binding domain have been deleted can also be constructed, totarget PA to specific cell types. Although the foregoing and the priorart describe specific deletion and structure-function analysis of PA,any biologically active form of PA can be used in the present invention.

Amino-terminal residues 1-254 of LF are sufficient for PA bindingactivity. Amino acid residues 199-253 may not all be required for PAbinding activity. One embodiment of LF is amino acids 1-254 of nativeLF. Any embodiment that contains at least about amino acids 1-254 ofnative LF can be used in the present invention, for example, native LF.Nontoxic embodiments of LF are preferred.

PA and LF fusion proteins can be produced using recombinant nucleicacids that encode a single-chain fusion proteins. The fusion protein canbe expressed as a single chain using in vivo or in vitro biologicalsystems. Using current methods of chemical synthesis, compounds can bealso be chemically bound to PA or LF. The fusion protein can be testedempirically for receptor binding, PA or LF binding, and internalizationfollowing the methods set forth in the Examples.

In addition, functional groups capable of forming covalent bonds withthe amino- and carboxyl-terminal amino acids or side groups of aminoacids are well known to those of skill in the art. For example,functional groups capable of binding the terminal amino group includeanhydrides, carbodiimides, acid chlorides, and activated esters.Similarly, function-al groups capable of forming covalent linkages withthe terminal carboxyl include amines and alcohols. Such functionalgroups can be used to bind compound to LF at either the amino- orcarboxyl-terminus. Compound can also be bound to LF through interactionsof amino acid residue side groups, such as the SH group of cysteine(see, e.g., Thorpe et al., Monoclonal Antibody-Toxin Conjugates: Aimingthe Magic Bullet, in Monoclonal Antibodies in Clinical Medicine, pp.168-190 (1982); Waldmann, Science, 252: 1657 (1991); U.S. Pat. Nos.4,545,985 and 4,894,443). The procedure for attaching an agent to anantibody or other polypeptide targeting molecule will vary according tothe chemical structure of the agent. As example, a cysteine residue canadded at the end of LF. Since there are no other cysteines in LF, thissingle cysteine provides a convenient attachment point through which tochemically conjugate other proteins through disulfide bonds. Althoughcertain of the methods of the invention have been described as using LFfusion proteins, it will be understood that other LF compositions havingchemically attached compounds can be used in the methods of theinvention.

Protective antigen proteins can be produced from nucleic acid constructsencoding mutants, in which the naturally occurring furin cleavage sitehas been replaced by an MMP or a plasminogen activator cleavage site. Inaddition, LF proteins, and LF and PA fusion proteins can also beexpressed from nucleic acid constructs according to standardmethodology. Those of skill in the art will recognize a wide variety ofways to introduce mutations into a nucleic acid encoding protectiveantigen or to construct a mutant protective antigen-encoding nucleicacid. Such methods are well known in the art (see Sambrook et al.,Molecular Cloning, A Laboratory Manual (2^(nd) ed. 1989); Kriegler, GeneTransfer and Expression: A Laboratory Manual (1990); and CurrentProtocols in Molecular Biology (Ausubel et al., eds., 1994)). In someembodiments, nucleic acids of the invention are generated using PCR(see, e.g., Examples I and III). For example, using overlap PCRprotective antigen encoding nucleic acids can be generated bysubstituting the nucleic acid subsequence that encodes the furin sitewith a nucleic acid subsequence that encodes a matrix metalloproteinase(MMP) site (e.g., GPLGMLSQ and GPLGLWAQ; SEQ ID NOS:2 ands 3) (see,e.g., Example I). Similarly, an overlap PCR method can be used toconstruct the protective antigen proteins in which the furin site isreplaced by a plasminogen activator cleavage site (e.g., the uPA and tPAphysiological substrate sequence PCPGRVVGG (SEQ ID NO:4), the uPAfavorite sequence PGSGRSA (SEQ ID NO:5), the uPA favorite sequencePGSGKSA (SEQ ID NO:6), or the tPA favorite sequence PQRGRSA (SEQ IDNO:7)) (see, e.g., Example III).

B. Expression of LF, PA and LF and PA Fusion Proteins

To obtain high level expression of a nucleic acid (e.g., cDNA, genomicDNA, PCR product, etc. or combinations thereof) encoding a native (e.g.,PA) or mutant protective antigen protein (e.g., PA-L1, PA-L2, PA-U1,PA-U2, PA-U3, PA-U4, etc.), LF, or a PA or LF fusion protein, onetypically subclones the protective antigen encoding nucleic acid into anexpression vector that contains a strong promoter to directtranscription, a transcription/translation terminator, and if for anucleic acid encoding a protein, a ribosome binding site fortranslational initiation. Suitable bacterial promoters are well known inthe art and described, e.g., in Sambrook et al. and Ausubel et al.Bacterial expression systems for expressing the protective antigenencoding nucleic acid are available in, e.g., E. coli, Bacillus sp., andSalmonella (Palva et al., Gene 22:229-235 (1983)). Kits for suchexpression systems are commercially available. Eukaryotic expressionsystems for mammalian cells, yeast, and insect cells are well known inthe art and are also commercially available.

In some embodiment, protective antigen containing proteins are expressedin non-virulent strains of Bacillus using Bacillus expression plasmidscontaining nucleic acid sequences encoding the particular protectiveantigen protein (see, e.g., Singh, Y., et al., J Biol Chem,264:19103-19107 (1989)). The protective antigen containing proteins canbe isolated from the Bacillus culture using protein purification methods(see, e.g., Varughese, M., et al., Infect Immun, 67:1860-1865 (1999)).

The promoter used to direct expression of a protective antigen encodingnucleic acid depends on the particular application. The promoter ispreferably positioned about the same distance from the heterologoustranscription start site as it is from the transcription start site inits natural setting. As is known in the art, however, some variation inthis distance can be accommodated without loss of promoter function. Thepromoter typically can also include elements that are responsive totransactivation, e.g., Gal4 responsive elements, lac repressorresponsive elements, and the like. The promoter can be constitutive orinducible, heterologous or homologous.

In addition to the promoter, the expression vector typically contains atranscription unit or expression cassette that contains all theadditional elements required for the expression of the nucleic acid inhost cells. A typical expression cassette thus contains a promoteroperably linked, e.g., to the nucleic acid sequence encoding theprotective antigen containing protein, and signals required forefficient expression and termination and processing of the transcript,ribosome binding sites, and translation termination. The nucleic acidsequence may typically be linked to a cleavable signal peptide sequenceto promote secretion of the encoded protein by the transformed cell.Such signal peptides would include, among others, the signal peptidesfrom bacterial proteins, or mammalian proteins such as tissueplasminogen activator, insulin, and neuron growth factor, and juvenilehormone esterase of Heliothis virescens. Additional elements of thecassette may include enhancers and, if genomic DNA is used as thestructural gene, introns with functional splice donor and acceptorsites.

In addition to a promoter sequence, the expression cassette should alsocontain a transcription termination region downstream of the structuralgene to provide for efficient termination and processing, if desired.The termination region may be obtained from the same gene as thepromoter sequence or may be obtained from different genes.

The particular expression vector used to transport the geneticinformation into the cell is not particularly critical. Any of theconventional vectors used for expression in eukaryotic or prokaryoticcells may be used. Standard bacterial expression vectors includeplasmids such as pBR322 based plasmids, pSKF, pET23D, and fusionexpression systems such as GST and LacZ. Epitope tags can also be addedto recombinant proteins to provide convenient methods of isolation,e.g., c-myc.

Expression vectors containing regulatory elements from eukaryoticviruses are typically used in eukaryotic expression vectors, e.g., SV40vectors, papilloma virus vectors, and vectors derived from Epstein-Barrvirus. Other exemplary eukaryotic vectors include pMSG, pAV009/A+,pMT010/A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowingexpression of proteins under the direction of the SV40 early promoter,SV40 later promoter, metallothionein promoter, murine mammary tumorvirus promoter, Rous sarcoma virus promoter, polyhedrin promoter, orother promoters shown effective for expression in eukaryotic cells.

Some expression systems have markers that provide gene amplificationsuch as thymidine kinase, hygromycin B phosphotransferase, anddihydrofolate reductase. Alternatively, high yield expression systemsnot involving gene amplification are also suitable, such as using abaculovirus vector in insect cells, with a protective antigen encodingnucleic acid under the direction of the polyhedrin promoter or otherstrong baculovirus promoters.

The elements that are typically included in expression vectors alsoinclude a replicon that functions in E. coli, a gene encoding antibioticresistance to permit selection of bacteria that harbor recombinantplasmids, and unique restriction sites in nonessential regions of theplasmid to allow insertion of heterologous sequences. The particularantibiotic resistance gene chosen is not critical, any of the manyresistance genes known in the art are suitable. The prokaryoticsequences are preferably chosen such that they do not interfere with thereplication of the DNA in eukaryotic cells, if necessary.

Standard transfection methods are used to produce bacterial, mammalian,yeast or insect cell lines that express large quantities of protein,which are then purified using standard techniques (see, e.g., Colley etal., J Biol. Chem. 264:17619-17622 (1989); Guide to ProteinPurification, in Methods in Enzymology, vol. 182 (Deutscher, ed.,1990)). Transformation of eukaryotic and prokaryotic cells are performedaccording to standard techniques (see, e.g., Morrison, J. Bact.132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology101:347-362 (Wu et al., eds, 1983).

Any of the well known procedures for introducing foreign nucleotidesequences into host cells may be used. These include the use of calciumphosphate transfection, polybrene, protoplast fusion, electroporation,liposomes, microinjection, plasma vectors, viral vectors and any of theother well known methods for introducing cloned genomic DNA, cDNA,synthetic DNA or other foreign genetic material into a host cell (see,e.g., Sambrook et al., supra). It is only necessary that the particulargenetic engineering procedure used be capable of successfullyintroducing at least one gene into the host cell capable of expressingthe protein of choice.

After the expression vector is introduced into the cells, thetransfected cells are cultured under conditions favoring expression ofthe protective antigen containing protein, which is recovered from theculture using standard techniques identified below.

III. Purification of Polypeptides of the Invention

Recombinant proteins of the invention can be purified from any suitableexpression system, e.g., by expressing the proteins in B. anthracis andthen purifying the recombinant protein via conventional purificationtechniques (e.g., ammonium sulfate precipitation, ion exchangechromatography, gel filtration, etc.) and/or affinity purification,e.g., by using antibodies that recognize a specific epitope on theprotein or on part of the fusion protein, or by using glutathioneaffinity gel, which binds to GST (see, e.g., Scopes, ProteinPurification: Principles and Practice (1982); U.S. Pat. No. 4,673,641;Ausubel et al., supra; and Sambrook et al., supra). In some embodiments,the recombinant protein is a fusion protein with GST or Gal4 at theN-terminus. Those of skill in the art will recognize a wide variety ofpeptides and proteins that can be fused to the protective antigencontaining protein to facilitate purification (e.g., maltose bindingprotein, a polyhistidine peptide, etc.).

A. Purification of Proteins from Recombinant Bacteria

Recombinant and native proteins can be expressed by transformed bacteriain large amounts, typically after promoter induction; but expression canbe constitutive. Promoter induction with IPTG is one example of aninducible promoter system. Bacteria are grown according to standardprocedures in the art. Fresh or frozen bacteria cells are used forisolation of protein.

Proteins expressed in bacteria may form insoluble aggregates (“inclusionbodies”). Several protocols are suitable for purification of inclusionbodies. For example, purification of inclusion bodies typically involvesthe extraction, separation and/or purification of inclusion bodies bydisruption of bacterial cells, e.g., by incubation in a buffer of 50 mMTris/HCl pH 7.5, 50 mM NaCl, 5 mM MgCl₂, 1 mM DTT, 0.1 mM ATP, and 1 mMPMSF. The cell suspension can be lysed using 2-3 passages through aFrench press, homogenized using a Polytron (Brinkman Instruments) orsonicated on ice. Alternate methods of lysing bacteria are apparent tothose of skill in the art (see, e.g., Sambrook et al., supra; Ausubel etal., supra).

If necessary, the inclusion bodies are solubilized, and the lysed cellsuspension is typically centrifuged to remove unwanted insoluble matter.Proteins that formed the inclusion bodies may be renatured by dilutionor dialysis with a compatible buffer. Suitable solvents include, but arenot limited to urea (from about 4 M to about 8 M), formamide (at leastabout 80%, volume/volume basis), and guanidine hydrochloride (from about4 M to about 8 M). Some solvents which are capable of solubilizingaggregate-forming proteins, for example SDS (sodium dodecyl sulfate),70% formic acid, are inappropriate for use in this procedure due to thepossibility of irreversible denaturation of the proteins, accompanied bya lack of immunogenicity and/or activity. Although guanidinehydrochloride and similar agents are denaturants, this denaturation isnot irreversible and renaturation may occur upon removal (by dialysis,for example) or dilution of the denaturant, allowing re-formation ofimmunologically and/or biologically active protein. Other suitablebuffers are known to those skilled in the art. The protein of choice isseparated from other bacterial proteins by standard separationtechniques, e.g., ion exchange chromatography, ammonium sulfatefractionation, etc.

B. Standard Protein Separation Techniques for Purifying Proteins of theInvention

Solubility Fractionation

Often as an initial step, particularly if the protein mixture iscomplex, an initial salt fractionation can separate many of the unwantedhost cell proteins (or proteins derived from the cell culture media)from the recombinant protein of interest. The preferred salt is ammoniumsulfate. Ammonium sulfate precipitates proteins by effectively reducingthe amount of water in the protein mixture. Proteins then precipitate onthe basis of their solubility. The more hydrophobic a protein is, themore likely it is to precipitate at lower ammonium sulfateconcentrations. A typical protocol includes adding saturated ammoniumsulfate to a protein solution so that the resultant ammonium sulfateconcentration is between 20-30%. This concentration will precipitate themost hydrophobic of proteins. The precipitate is then discarded (unlessthe protein of interest is hydrophobic) and ammonium sulfate is added tothe supernatant to a concentration known to precipitate the protein ofinterest. Alternatively, the protein of interest in the supernatant canbe further purified using standard protein purification techniques. Theprecipitate is then solubilized in buffer and the excess salt removed ifnecessary, either through dialysis or diafiltration. Other methods thatrely on solubility of proteins, such as cold ethanol precipitation, arewell known to those of skill in the art and can be used to fractionatecomplex protein mixtures.

Size Differential Filtration

The molecular weight of the protein, e.g., PA-U1, etc., can be used toisolated the protein from proteins of greater and lesser size usingultrafiltration through membranes of different pore size (for example,Amicon or Millipore membranes). As a first step, the protein mixture isultrafiltered through a membrane with a pore size that has a lowermolecular weight cut-off than the molecular weight of the protein ofinterest. The retentate of the ultrafiltration is then ultrafilteredagainst a membrane with a molecular cut off greater than the molecularweight of the protein of interest. The recombinant protein will passthrough the membrane into the filtrate. The filtrate can then bechromatographed as described below.

Column Chromatography

The protein of choice can also be separated from other proteins on thebasis of its size, net surface charge, hydrophobicity, and affinity forligands. In addition, antibodies raised against proteins can beconjugated to column matrices and the proteins immunopurified. All ofthese methods are well known in the art. It will be apparent to one ofskill that chromatographic techniques can be performed at any scale andusing equipment from many different manufacturers (e.g., PharmaciaBiotech).

In some embodiments, the proteins are purified from culture supernatantsof Bacillus (see, e.g., Examples I and III). Briefly, the proteins arepurified by making a culture supernatant 5 mM in EDTA, 35% saturated inammonium sulfate and 1% in phenyl-Sepharose Fast Flow (Pharmacia). Thephenyl-Sepharose Fast Flow is then agitated and collected. The collectedresin is washed with 35% saturated ammonium sulfate and the protectiveantigens were then eluted with 10 mM HEPES-1 mM EDTA (pH 7.5). Theproteins can then be further purified using a MonoQ column (PharmaciaBiotech). The proteins can be eluted using a NaCl gradient in 10 mM CHES(2-[N-cyclohexylamino]ethanesulfonic acid)-0.06% (vol/vol) ethanolamine(pH 9.1). The pooled MonoQ fractions can then be dialyzed against thebuffer of choice for subsequent analysis or applications.

IV. Assays for Measuring Changes in Cell Growth

The administration of a functional PA and LF combination of theinvention to a cell can inhibit cellular proliferation of certain celltypes that overexpress MMPs and proteins of the plasminogen activationsystem, e.g., cancer cells, cells involved in inflammation, and thelike. One of skill in the art can readily identify functional proteinsand cells using methods that are well known in the art. Changes in cellgrowth can be assessed by using a variety of in vitro and in vivoassays, e.g., MTT assay, ability to grow on soft agar, changes incontact inhibition and density limitation of growth, changes in growthfactor or serum dependence, changes in the level of tumor specificmarkers, changes in invasiveness into Matrigel, changes in cell cyclepattern, changes in tumor growth in vivo, such as in transgenic mice,etc.

The term “over-expressing” refers to a cell that expresses a matrixmetalloproteinase, a plasminogen activator or a plasminogen activatorreceptor mRNA or protein in amounts at least about twice that normallyproduced in a reference normal cell type, e.g., a Vero cell.Overexpression can result, e.g., from selective pressure in culturemedia, transformation, activation of endogenous genes, or by addition ofexogenous genes. Overexpression can be analyzed using a variety ofassays known to those of skill in the art to determine if the gene orprotein is being overexpressed (e.g., northerns, RT-PCR, westerns,immunoassays, cytotoxicity assays, growth inhibition assays, enzymeassays, gelatin zymography, etc.). An example of a cell overexpressing amatrix metalloproteinase are the tumor cell lines, fibrosarcoma HT1080,melanoma A2058 and breast cancer MDA-MB-231. An example of a cell whichdoes not overexpress a matrix metalloproteinase is the non-tumor cellline Vero. An example of a cells that overexpress a plasminogenactivator receptor are the uPAR overexpressing cell types Hela, A2058,and Bowes. An example of a cell which does not overexpress a plasminogenactivator receptor is the non-tumor cell line Vero. An example of acells that overexpress a tissue-type plasminogen activator are celltypes human melanoma Bowes and human primary vascular endothelial cells.An example of a cell which does not overexpress a plasminogen activatorreceptor is the non-tumor cell line Vero.

A. Assays for Changes in Cell Growth by Administration of ProtectiveAntigen and Lethal Factor

One or more of the following assays can be used to identify proteins ofthe invention which are capable of regulating cell proliferation. Thephrase “protective antigen constructs” refers to a protective antigenprotein of the invention. Functional protective antigen constructsidentified by the following assays can then be used to treat disease andconditions, e.g., to inhibit abnormal cellular proliferation andtransformation. Thus, these assays can be sued to identify protectiveantigen proteins that are useful in conjunction with lethal factorcontaining proteins to inhibit cell growth of tumors, cancers, cancerouscells, and other pathogenic cell types.

Soft Agar Growth or Colony Formation in Suspension

Soft agar growth or colony formation in suspension assays can be used toidentify protective antigen constructs, which when used in conjunctionwith a LF construct, inhibit abnormal cellular proliferation andtransformation. Typically, transformed host cells (e.g., cells that growon soft agar) are used in this assay. Techniques for soft agar growth orcolony formation in suspension assays are described in Freshney, Cultureof Animal Cells a Manual of Basic Technique, 3^(rd) ed., Wiley-Liss, NewYork (1994), herein incorporated by reference. See also, the methodssection of Garkavtsev et al. (1996), supra, herein incorporated byreference.

Normal cells require a solid substrate to attach and grow. When thecells are transformed, they lose this phenotype and grow detached fromthe substrate. For example, transformed cells can grow in stirredsuspension culture or suspended in semi-solid media, such as semi-solidor soft agar. The transformed cells, when transfected with tumorsuppressor genes, regenerate normal phenotype and require a solidsubstrate to attach and grow.

Administration of an active protective antigen protein and an active LFcontaining protein to transformed cells would reduce or eliminate thehost cells' ability to grow in stirred suspension culture or suspendedin semi-solid media, such as semi-solid or soft. This is because thetransformed cells would regenerate anchorage dependence of normal cells,and therefore require a solid substrate to grow. Therefore, this assaycan be used to identify protective antigen constructs that can functionwith a lethal factor protein to inhibit cell growth. Once identified,such protective antigen constructs can be used in a number of diagnosticor therapeutic methods, e.g., in cancer therapy to inhibit abnormalcellular proliferation and transformation.

Contact Inhibition and Density Limitation of Growth

Contact inhibition and density limitation of growth assays can be usedto identify protective antigen constructs which are capable ofinhibiting abnormal proliferation and transformation in host cells.Typically, transformed host cells (e.g., cells that are not contactinhibited) are used in this assay. Administration of a protectiveantigen construct and a lethal factor construct to these transformedhost cells would result in cells which are contact inhibited and grow toa lower saturation density than the transformed cells. Therefore, thisassay can be used to identify protective antigen constructs which areuseful in compositions for inhibiting cell growth. Once identified, suchprotective antigen constructs can be used in disease therapy to inhibitabnormal cellular proliferation and transformation.

Alternatively, labeling index with [³H]-thymidine at saturation densitycan be used to measure density limitation of growth. See Freshney(1994), supra. The transformed cells, when treated with a functionalPA/LF combination, regenerate a normal phenotype and become contactinhibited and would grow to a lower density. In this assay, labelingindex with [³H]-thymidine at saturation density is a preferred method ofmeasuring density limitation of growth. Transformed host cells aretreated with a protective antigen construct and a lethal factorconstruct (e.g., LP59) and are grown for 24 hours at saturation densityin non-limiting medium conditions. The percentage of cells labeling with[³H]-thymidine is determined autoradiographically. See, Freshney (1994),supra. The host cells treated with a functional protective antigenconstruct would give arise to a lower labeling index compared to control(e.g., transformed host cells treated with a non-functional protectiveantigen construct or non-functional lethal factor construct).

Growth Factor or Serum Dependence

Growth factor or serum dependence can be used as an assay to identifyfunctional protective antigen constructs. Transformed cells have a lowerserum dependence than their normal counterparts (see, e.g., Temin, J.Natl. Cancer Insti. 37:167-175 (1966); Eagle et al., J. Exp. Med.131:836-879 (1970)); Freshney, supra. This is in part due to release ofvarious growth factors by the transformed cells. When a tumor suppressorgene is transfected and expressed in these transformed cells, the cellswould reacquire serum dependence and would release growth factors at alower level. Therefore, this assay can be used to identify protectiveantigen constructs which are able to act in conjunction with a lethalfactor to inhibit cell growth. Growth factor or serum dependence oftransformed host cells which are transfected with a protective antigenconstruct can be compared with that of control (e.g., transformed hostcells which are treated with a non-functional protective antigen ornon-functional lethal factor). Transformed host cells treated with afunctional protective antigen would exhibit an increase in growth factorand serum dependence compared to control.

Tumor Specific Markers Levels

Tumor cells release an increased amount of certain factors (hereinafter“tumor specific markers”) than their normal counterparts. For example,tumor angiogenesis factor (TAF) is released at a higher level in tumorcells than their normal counterparts. See, e.g., Folkman, Angiogenesisand cancer, Sem Cancer Biol. (1992)).

Tumor specific markers can be assayed for to identify protective antigenconstructs, which when administered with a lethal factor construct,decrease the level of release of these markers from host cells.Typically, transformed or tumorigenic host cells are used.Administration of a protective antigen and a lethal factor to these hostcells would reduce or eliminate the release of tumor specific markersfrom these cells. Therefore, this assay can be used to identifyprotective antigen constructs are functional in suppressing tumors.

Various techniques which measure the release of these factors aredescribed in Freshney (1994), supra. Also, see, Unkless et al., J. Biol.Chem. 249:4295-4305 (1974); Strickland & Beers, J. Biol. Chem.251:5694-5702 (1976); Whur et al., Br. J. Cancer 42:305-312 (1980);Gulino, Angiogenesis, tumor vascularization, and potential interferencewith tumor growth. In Mihich, E. (ed): “Biological Responses in Cancer.”New York, Plenum (1985); Freshney Anticancer Res. 5:111-130 (1985).

Cytotoxicity Assay with MTT

The cytotoxicity of a particular PA/LF combination can also be assayedusing the MTT cytotoxicity assay. Cells are seeded and grown to 80 to100% confluence. The cells are then were washed twice with serum-freeDMEM to remove residual FCS and contacted with a particular PA/LFcombination. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide) is then added to the cells and oxidized MTT (indicative of alive cell) is solubilized and quantified.

Invasiveness into Matrigel

The degree of invasiveness into Matrigel or some other extracellularmatrix constituent can be used as an assay to identify protectiveantigen constructs which are capable of inhibiting abnormal cellproliferation and tumor growth. Tumor cells exhibit a good correlationbetween malignancy and invasiveness of cells into Matrigel or some otherextracellular matrix constituent. In this assay, tumorigenic cells aretypically used. Administration of an active protective antigen/lethalfactor protein combination to these tumorigenic host cells woulddecrease their invasiveness. Therefore, functional protective antigenconstructs can be identified by measuring changes in the level ofinvasiveness between the tumorigenic cells before and after theadministration of the protective antigen and lethal factor constructs.

Techniques described in Freshney (1994), supra, can be used. Briefly,the level of invasion of tumorigenic cells can be measured by usingfilters coated with Matrigel or some other extracellular matrixconstituent. Penetration into the gel, or through to the distal side ofthe filter, is rated as invasiveness, and rated histologically by numberof cells and distance moved, or by prelabeling the cells with ¹²⁵I andcounting the radioactivity on the distal side of the filter or bottom ofthe dish. See, e.g., Freshney (1984), supra.

G₀/G₁ Cell Cycle Arrest Analysis

G₀/G₁ cell cycle arrest can be used as an assay to identify functionalprotective antigen construct. PA/LF construct administration can causeG1 cell cycle arrest. In this assay, cell lines can be used to screenfor functional protective antigen constructs. Cells are treated with aputative protective antigen construct and a lethal factor construct. Thecells can be transfected with a nucleic acid comprising a marker gene,such as a gene that encodes green fluorescent protein. Administration ofa functional protective antigen/lethal factor combination would causeG₀/G₁ cell cycle arrest. Methods known in the art can be used to measurethe degree of G₁ cell cycle arrest. For example, the propidium iodidesignal can be used as a measure for DNA content to determine cell cycleprofiles on a flow cytometer. The percent of the cells in each cellcycle can be calculated. Cells exposed to a functional protectiveantigen would exhibit a higher number of cells that are arrested inG₀/G₁ phase compared to control (e.g., treated in the absence of aprotective antigen).

Tumor Growth In Vivo

Effects of PA/LF on cell growth can be tested in transgenic orimmune-suppressed mice. Transgenic mice can be made, in which a tumorsuppressor is disrupted (knock-out mice) or a tumor promoting gene isoverexpressed. Such mice can be used to study effects of protectiveantigen as a method of inhibiting tumors in vivo.

Knock-out transgenic mice can be made by insertion of a marker gene orother heterologous gene into a tumor suppressor gene site in the mousegenome via homologous recombination. Such mice can also be made bysubstituting the endogenous tumor suppressor with a mutated version ofthe tumor suppressor gene, or by mutating the endogenous tumorsuppressor, e.g., by exposure to carcinogens.

A DNA construct is introduced into the nuclei of embryonic stem cells.Cells containing the newly engineered genetic lesion are injected into ahost mouse embryo, which is re-implanted into a recipient female. Someof these embryos develop into chimeric mice that possess germ cellspartially derived from the mutant cell line. Therefore, by breeding thechimeric mice it is possible to obtain a new line of mice containing theintroduced genetic lesion (see, e.g., Capecchi et al., Science 244:1288(1989)). Chimeric targeted mice can be derived according to Hogan etal., Manipulating the Mouse Embryo: A Laboratory Manual, Cold SpringHarbor Laboratory (1988) and Teratocarcinomas and Embryonic Stem Cells:A Practical Approach, Robertson, ed., IRL Press, Washington, D.C.,(1987).

These knock-out mice can be used as hosts to test the effects of variousprotective antigen constructs on cell growth. These transgenic mice witha tumor suppressor gene knocked out would develop abnormal cellproliferation and tumor growth. They can be used as hosts to test theeffects of various protective antigen constructs on cell growth. Forexample, introduction of protective antigen constructs and lethal factorconstructs into these knock-out mice would inhibit abnormal cellularproliferation and suppress tumor growth.

Alternatively, various immune-suppressed or immune-deficient hostanimals can be used. For example, genetically athymic “nude” mouse (see,e.g., Giovanella et al., J. Natl. Cancer Inst. 52:921 (1974)), a SCIDmouse, a thymectomized mouse, or an irradiated mouse (see, e.g., Bradleyet al., Br. J. Cancer 38:263 (1978); Selby et al., Br. J. Cancer 41:52(1980)) can be used as a host. Transplantable tumor cells (typicallyabout 10⁶ cells) injected into isogenic hosts will produce invasivetumors in a high proportions of cases, while normal cells of similarorigin will not. In hosts which developed invasive tumors, cells areexposed to a protective antigen construct/lethal factor combination(e.g., by subcutaneous injection). After a suitable length of time,preferably 4-8 weeks, tumor growth is measured (e.g., by volume or byits two largest dimensions) and compared to the control. Tumors thathave statistically significant reduction (using, e.g., Student's T test)are said to have inhibited growth. Using reduction of tumor size as anassay, functional protective antigen constructs which are capable ofinhibiting abnormal cell proliferation can be identified. This model canalso be used to identify functional mutant versions of protectiveantigen.

V. Pharmaceutical Compositions Administration

Protective antigen containing proteins and lethal factor containingproteins can be administered directly to the patient, e.g., forinhibition of cancer, tumor, or precancer cells in vivo, etc.Administration is by any of the routes normally used for introducing acompound into ultimate contact with the tissue to be treated. Thecompounds are administered in any suitable manner, preferably withpharmaceutically acceptable carriers. Suitable methods of administeringsuch compounds are available and well known to those of skill in theart, and, although more than one route can be used to administer aparticular composition, a particular route can often provide a moreimmediate and more effective reaction than another route.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions of thepresent invention (see, e.g., Remington's Pharmaceutical Sciences,17^(th) ed. 1985)). For example, if in vivo delivery of a biologicallyactive protective antigen protein is desired, the methods described inSchwarze et al. (see Science 285:1569-1572 (1999)) can be used.

The compounds, alone or in combination with other suitable components,can be made into aerosol formulations (i.e., they can be “nebulized”) tobe administered via inhalation. Aerosol formulations can be placed intopressurized acceptable propellants, such as dichlorodifluoromethane,propane, nitrogen, and the like.

Formulations suitable for parenteral administration, such as, forexample, by intravenous, intramuscular, intradermal, and subcutaneousroutes, include aqueous and non-aqueous, isotonic sterile injectionsolutions, which can contain antioxidants, buffers, bacteriostats, andsolutes that render the formulation isotonic with the blood of theintended recipient, and aqueous and non-aqueous sterile suspensions thatcan include suspending agents, solubilizers, thickening agents,stabilizers, and preservatives. In the practice of this invention,compositions can be administered, for example, by intravenous infusion,orally, topically, intraperitoneally, intravesically or intrathecally.The formulations of compounds can be presented in unit-dose ormulti-dose sealed containers, such as ampules and vials. Injectionsolutions and suspensions can be prepared from sterile powders,granules, and tablets of the kind previously described.

The dose administered to a patient (“a therapeutically effectiveamount”), in the context of the present invention should be sufficientto effect a beneficial therapeutic response in the patient over time.The dose will be determined by the efficacy of the particular compoundemployed and the condition of the patient, as well as the body weight orsurface area of the patient to be treated. The size of the dose alsowill be determined by the existence, nature, and extent of any adverseside-effects that accompany the administration of a particular compoundor vector in a particular patient

In determining the effective amount of the compound(s) to beadministered in the treatment or prophylaxis of cancer, the physicianevaluates circulating plasma levels of the respective compound(s),progression of the disease, and the production of anti-compoundantibodies. In general, the dose equivalent of a compound is from about1 ng/kg to 10 mg/kg for a typical patient. Administration of compoundsis well known to those of skill in the art (see, e.g., Bansinath et al.,Neurochem Res. 18:1063-1066 (1993); Iwasaki et al., Jpn. J. Cancer Res.88:861-866 (1997); Tabrizi-Rad et al., Br. J. Pharmacol. 111:394-396(1994)).

For administration, compounds of the present invention can beadministered at a rate determined by the LD-50 of the particularcompound, and its side-effects at various concentrations, as applied tothe mass and overall health of the patient. Administration can beaccomplished via single or divided doses.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

EXAMPLES

The following examples are provided by way of illustration only and notby way of limitation. Those of skill in the art will readily recognize avariety of noncritical parameters that could be changed or modified toyield essentially similar results.

Example I Construction of Mutant PA with Matrix MetalloproteinaseCleavage Sites

A. Materials

Enzymes for DNA manipulation and modification were purchased from NewEngland Biolabs (Beverly, Mass.). FP59 and soluble form furin wereprepared in our laboratory according to standard methodology. ActiveMMP-2 was a kind gift from Dr. William Stetler-Stevenson, active formMMP-9 was purchased from CALBIOCHEM (San Diego, Calif.). MMP inhibitorsBB-94 (Batimastat) and BB-2516 (Marimastat) were kind gifts from BritishBiotechnology Limited, GM6001 was a kind gift from Dr. Richard E.Galardy prepared as described (Grobelny, D., et al., Biochemistry,31:7152-7154 (1992)). Rabbit anti-PA polyclonal antibody (#5308) wasmade in our laboratory. Rabbit anti-MT-MMP1 (AB815) was purchased fromCHEMICON International, Inc. (Temecula, Calif.). The sequence for LF canbe found, e.g., in Robertson & Leppla, Gene 44: 71-78 (1986). Thesequence for PA is described, e.g., in Singh et al., J. Biol. Chem. 264:19103-19107 (1989) (expression vector pYS5); Leppla, in Methods inEnzymology, vol. 165, pp. 103-116 (Harshman ed., 1988). Site-directedmutagenesis of PA molecules has been previously described (Singh et al.,J. Biol. Chem. 269: 29039-29046 (1994)

Construction of PA MMP Substrate Mutants

Overlap PCR was used to construct the PA mutants with the furin sitereplaced by MMP substrate octapeptide GPLGMLSQ (SEQ ID NO:2) in PA-L1and GPLGLWAQ (SEQ ID NO:3) in PA-L2. Wild type PA (WT-PA) expressionplasmid pYS5 (Singh, Y., et al., J Biol Chem, 264:19103-19107 (1989))was used as template. We used 5′ primer F (AAAGGAGAACGTATATGA (SEQ IDNO:8), underlined are SD sequence and start codon of PA) and thephosphorylated primer R1 (pTGAGTTCGAAGATTTTTGTTTTAATTCTGG (SEQ ID NO:9),annealing to the sequence corresponding to P₁₅₄-S₁₆₃) to amplify thefragment N. We used the mutagenic phosphorylated primer H1(pGGACCATTAGGAATGTGGAGTCAAAGTA CAAGTGCTGGACCTACGGTTCCG (SEQ ID NO:10),encoding MMP substrate GPLGMLSQ (SEQ ID NO:2) and S₁₆₈-P₁₇₆) and reverseprimer R2 ACGTTTATCTCTTATTAAAAT (SEQ ID NO:11), annealing to thesequence compassing I₅₈₉-R₅₉₅) to amplify the mutagenic fragment M1. Weused a phosphorylated mutagenic primer H2 (pGGACCATTAGGATTATGGGCACAAAGTACAAGTGCTGGACCTACGGTTCCG (SEQ ID NO:12), encoding MMP substrateGPLGLWAQ (SEQ ID NO:3) and S₁₆₈-P₁₇₆) to amplify mutagenic fragment M2.Then used primer F and R2 to amplify the ligation products of N and M1,N and M2, respectively, resulting in the mutagenic fragments L1 and L2,in which the coding sequence for furin site (RKKR₁₆₇; SEQ ID NO:1) werereplaced by MMP substrate sequence GPLGMLSQ and GPLGLWAQ (SEQ ID NOS:2and 3), respectively. The HindIII/PstI digests of L1 and L2, whichincluded the mutation sites, were cloned between HindIII and PstI siteof pYS5. The resulting expression plasmids were named pYS-PA-L1 andpYS-PA-L2, their expression products, the PA mutated proteins, wereaccordingly named PA-L1 and PA-L2.

Expression and Purification of WT-PA, PA-L1 and PA-L2

To express WT-PA, PA-L1 and PA-L2, expression plasmids pYS5, pYS-PA-L1and pYS-PA-L2 were transformed into non-virulent strain B. anthracisUM23C1-1, and grown in FA medium (Singh, Y., et al., J Biol Chem,264:19103-19107 (1989)) with 20 μg/ml of kanamycin for 16 h at 37° C.,PA proteins were purified by ammonium sulfate precipitation followed bymonoQ column (Pharmacia Biotech) chromatography, as described previously(Varughese, M., et al., Infect Immun, 67:1860-1865 (1999)).

In Vitro Cleavage of WT-PA, PA-L1 and PA-L2 by Furin, MMP-2 and MMP-9

To test whether PA-L1 and PA-L2 had the ability to be processed by MMP-2and MMP-9 rather than furin, in vitro cleavage of WT-PA, PA-L1 and PA-L2were performed. For furin cleavage, 50 μl volume of reaction in PBS, pH7.4, 25 mM HEPES, 0.2 mM EDTA, 0.2 mM EGTA, 100 μg/ml ovalbumin, 1.0 mMCaCl₂, 1.0 mM MgCl₂, including 5 μs of WT-PA, PA-L1 and PA-L2,respectively. Digestion was started by addition 0.1 μg of soluble formof furin and incubated at 37° C., aliquots (5 μl) were withdrawn atdifferent time points. Cleavage was detected by western blotting with arabbit anti-PA antibody. For western blotting, the sample aliquots wereseparated by PAGE using 10-20% gradient Tris-glycine gel (Novex, SanDiego, Calif.) and electroblotted to a nitrocellulose membrane (Novex,San Diego, Calif.). The membrane was blocked with 5% (w/v) non-fat milkand hybridized by using rabbit anti-PA polyclonal antibody (#5308). Blotwas washed and incubated with an HRP-conjugated goat anti-rabbitantibody (sc-2004, Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.)and was visualized by TMB Stabilized Substrate for HRP (Promega,Madison, Wis.). For MMP-2 and MMP-9 cleavage, 5 μg each of WT-PA, PA-L1and PA-L2 was incubated with 0.2 μg active MMP-2 or 0.2 μg active MMP-9,respectively, in 50 μl of reactions including 50 mM HEPES, pH 7.5, 10 mMCaCl₂, 200 mM NaCl, 0.05% (v/v) Brij-35 and 50 μM ZnSO₄. Aliquots (5 μl)were withdrawn at different time points and were analyzed by westernblotting with rabbit anti-PA polyclonal antibody (#5308) as describedabove.

Cells and Culture Medium

Vero cells, COS-7 cells, human fibrosarcoma HT1080 cells, human melanomaA2058 cells and human breast cancer MDA-MB-231 cells were obtained fromATCC (Rockville, Md.). All cells were grown in Dulbecco' ModifiedEagle's Medium (DMEM) with 0.45% glucose, 10% fetal bovine serum, 2 mMglutamine. Cells were maintained at 37° C. in a 5% CO₂ incubator. Cellswere dissociated with a solution of 0.05% trypsin, 0.02% EDTA, 0.01 Msodium phosphate, pH 7.4, and were usually subcultured at a split ratioof 1:4.

Preparation of Cell Extracts and Condition Media for Gelatin Zymography

Cells were cultured in 75 cm² flask to 80-100% of confluence at 37° C.in DMEM supplemented with 10% FCS. Then the cells were washed twice withserum-free DMEM to remove residual FCS, and lysed for 10 min on ice with1 ml/flask of 0.5% (v/v) Triton X-100 in 0.1 M Tris-HCl, pH 8.0, andscraped with a rubber policeman. The cell lysates were centrifuged at10,000 rpm for 10 min at 4° C., the concentrations of the proteins weredetermined by BCA Protein Assay Kit (PIERCE, Rockford, Ill.), and wasadjusted to 1 mg/ml by lysis buffer. For collection the conditionedmedia, the cells were incubated for 24 h with 4 ml/flask of serum-freeDMEM. The culture supernatants were harvested, and cellular debrisremoved by centrifugation at 10,000 rpm for 10 min at 4° C. Cell lysatesand conditioned media were frozen at −70° C. or immediately processedfor zymographic analysis.

Gelatin Zymography

Cell extracts (1 ml) or conditioned media normalized to proteinconcentrations of the corresponding cell extract (3-4 ml) were incubatedat 4° C. for 1 h in an end-over-end mixer with 50 μl ofgelatin-sepharose 4B (Pharmacia Biotech AB) equilibrated with 50 mMTris-HCl, 150 mM NaCl, 5 mM CaCl₂, 0.02% (v/v) Tween-20, 10 mM EDTA, pH7.5. After 4 washes with 1 ml of equilibration buffer containing 200 mMNaCl, the beads were resuspended in 30 μl 4× non-reducing sample buffer,centrifuged to collect the supernatants and loaded on 10% gelatinzymogram gel (Novex, San Diego, Calif.). After electrophoresis, the gelwas soaked in Renaturing Buffer (Novex, San Diego, Calif.) for twicewith 30 min each to renature gelatinases at room temperature. The gelwas then equilibrated in Developing Buffer (Novex, San Diego, Calif.),which added back a divalent metal cation required for enzymaticactivity, first for 30 min at room temperature and then in new buffer at37° C. for overnight. The gel was then stained overnight with 0.5% (w/v)Commassie Brilliant Blue R-250 in 45% (v/v) methanol, 10% acetic acidand destained in the same solution without dye.

Cytotoxicity Assay with MTT

Cytotoxicity of WT-PA, PA-L1 and PA-L2 to the test cells were performedin 96-well plates. Cells were properly seeded into 96-well plates sothat they reached 80 to 100% of confluence the next day. The cells werewashed twice with serum-free DMEM to remove residual FCS. Then seriallydiluted WT-PA, PA-L1 or PA-L2 (from 0 to 1000 ng/ml) combined with FP59(50 ng/ml) in serum-free DMEM were added to the cells to give a totalvolume of 200 μl/well. One group of cells was challenged with the toxinsfor 6 hours, then removed the toxins replaced with fresh DMEMsupplemented with 10% FCS. For the cytotoxic action of FP59 relies oninhibition of initial protein synthesis by ADP ribosylating EF-2 andusually need 24-48 hours to show the toxicity, cytotoxicity was allowedto develop for 48 hours. After that cell viability was assayed by adding50 μl of 2.5 mg/ml MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide). Thecells were incubated with MTT for 45 min at 37° C., live cells oxidizedMTT to blue dye precipitated in cytosol while dead cells remainedcolorless. Then removed media and solubilized the blue precipitate with100 μl/well of 0.5% (w/v) SDS, 25 mM HCl, in 90% (v/v) isopropanol. Theplates were vortexed and the intensity of the oxidized MTT read at 570nm using the microplate reader. Another group of cells was challengedwith the toxins for 48 hours in serum-free DMEM, then viability wasdetermined by cytotoxicity assay with MTT as described above

Cytotoxicity Assay in the Co-Culture Model

We designed a co-culture model to mimic the in vivo condition to verifywhether PA-L1 and PA-L2 specifically killed MMP expressing tumor cells,not MMP non-expressing cells. Vero, HT1080, A2058 and MDA-MB-231 cellswere cultured into the different chambers of 8-chamber slide (Nalge NuncInternational, Naperville, Ill.) to 80-100% of confluence. Then thecells were washed twice with serum-free DMEM, the chamber partition wasremoved, and the slide was put into a petri culture dish with serum freemedium, so that the different cells were in the same cultureenvironment. PA, PA-L1 or PA-L2 (300 ng/ml) each plus FP59 (50 ng/ml),or FP59 (50 ng/ml) alone were added to the cells and incubated to 48hours. Then MTT (0.5 mg/ml) was added for 45 min at 37° C., thepartition was remounted, the oxidized MTT was dissolved as describedabove to determine cell viability for each chamber.

Cell Binding and Processing Assay of WT-PA, PA-L1 and PA-L2

Binding and processing of WT-PA, PA-L1 and PA-L2 on the surface of Verocells and HT1080 cells was assayed. Vero and HT1080 cells were grown in24-well plate to 80-100% of confluence and washed twice with serum-freeDMEM to remove residual FCS. Then the cells were incubated with 1000ng/ml of WT-PA, PA-L1 and PA-L2, respectively, for different length oftime (0, 10 min, 40 min, 120 min and 360 min) at 37° C. in serum-freeDMEM. The cells were washed three times to remove unbound PA proteins.Cells were lysed in 100 μl/well modified RIPA lysis buffer (50 mMTris-HCl, pH 7.4, 1% NP40, 0.25 Na-deoxycholate, 150 mM NaCl, 1 mM EDTA,1 mM PMSF, 1 mg/ml each of aprotinin, leupeptin and pepstatin) on icefor 10 min. Equal amounts of protein from cell lysates were separated byPAGE using 10-20% gradient Tris-glycine gels (Novex, San Diego, Calif.).After transfer to nitrocellulose membranes, blocking was done with 5%non-fat milk. Western blotting used rabbit anti-PA polyclonal antibody(#5308). Blot was washed and incubated with an HRP-conjugated goatanti-rabbit antibody (sc-2004) (Santa Cruz Biotechnology, Inc., SantaCruz, Calif.) and was visualized by EL (PIERCE, Rockford, Ill.).

Construction and Transfection of MT1-MMP into COS-7 Cells

MT1-MMP cDNA was a generous gift of J. Windsor, AB. The pEGFPN1(Clontech Laboratories, Inc., Palo Alto, Calif.) mammalian expressionvector was used for fusing the C-terminus of MT1-MMP to the N-terminusof EGFP (red shifted variant of green fluorescent protein). The MT1-MMPcoding sequence was isolated with Tth III and then filled in with Pfuand inserted into the SmaI site of pEGFPN1. COS-7 Cells (2×10⁵ per dish)were transfected with expression vectors (2 μg) by means of SuperFect(10 ml) (Qiagen). Cells were incubated for 3 h. with the DNA-SuperFectcomplex in the presence of serum and antibiotic containing medium. Thecomplex containing medium was removed and cells grown in fresh serumcontaining medium for 48 h. Thereafter cells were grown in G418 (LifeTechnologies, Inc.) containing medium. Cells expressing the MT1-MMP/GFPfusion protein, named COSgMT1, were sorted from non-expressing cells byflowcytometry with a FACstar Plus (Becton Dickinson), excitation at 488nm.

B. Results

Generation of PA Mutants which can be Activated by MMPs

Crystal structure of PA showed that the furin cleavage site RKKR₁₆₇ isin the middle of a surface flexible, solvent exposed loop composed of aa162 to 175 (Petosa, C., et al., Nature, 385:833-838 (1997)). Cleavage inthis loop by furin-like proteases is essential to toxicity. To constructPA mutants specifically processed by MMPs, especially MMP-2 and MMP-9,instead of furin, the furin site RKKR₁₆₇ (SEQ ID NO:1) was replaced byMMP-2 and MMP-9 favorite sequences, GPLGMLSQ and GPLGLWAQ (SEQ ID NOS:2and 3), respectively, resulting in two PA mutants, PA-L1 and PA-L2 (FIG.1a ). These two MMP substrate octapeptides were designed based on thestudies of Netzel-Arneet et al (Netzel-Arnett, S., et al., J Biol Chem,266:6747-6755 (1991); Netzel-Arnett, S., et al., Biochemistry,32:6427-6432 (1993)), in which the sequence specificity of human MMP-2,MMP-9, matrilysin, MMP-1 and MMP-8 had been examined by measuring therate of hydrolysis of over 50 synthetic oligopeptides. These twooctapeptides are favorite substrates of MMP-2 and MMP-9, but alsooverlap to other MMP species (Netzel-Arnett, S., et al., J Biol Chem,266:6747-6755 (1991); Netzel-Arnett, S., et al., Biochemistry,32:6427-6432 (1993)). They are also potential substrates for MT1-MMP(Will, H., et al., J Biol Chem, 271:17119-17123 (1996)). PA-L1 and PA-L2coding sequences were constructed by overlap PCR, cloned into E.coli-Bacillus shuttle vector pYS5, and efficiently expressed innon-virulent Bacillus Anthracis UM23C1-1. The expression products weresecreted into the culture supernatants and reached to 20 to 50 mg/L.These two mutated PA proteins were roughly purified by ammonium sulfateprecipitation, followed by mono Q chromatography. The purified mutatedPA proteins PA-L1 and PA-L2 commiserated with WT-PA in SDS-PAGE, butmigrated faster than WT-PA in native gel because of the four positivelycharged residues RKKR (SEQ ID NO:1) of the furin site were replaced intonon-charged MMP octapeptides (data not shown).

To characterize WT-PA and these two PA mutants in susceptibility toproteases, they were subjected to the cleavage with soluble form furin,active form MMP-2 and MMP-9 in vitro. WT-PA was very sensitive to furin,but complete resistant to MMP-2 and MMP-9 (FIG. 1b ). In contrast, PA-L1and PA-L2 were completely resistant to furin, but got the new feature tobe efficiently processed into two fragments, PA63 and PA20, by MMP-2 andMMP-9 (FIGS. 1c and 1d ). There was no apparent difference between thetwo PA mutants in respect to the processing patterns by furin, MMP-2 andMMP-9. However, it seemed PA-L1 and PA-L2 were processed moreefficiently by MMP-2 than by MMP-9.

PA-L1 and PA-L2 Killed MMP Expressing Tumor Cells but not MMPNon-Expressing Cells

To test the hypothesis that PA-L1 and PA-L2 only kill MMP expressingtumor cells, but not MMP non-expressing normal cells, three human tumorcell lines, fibrosarcoma HT1080, melanoma A2058 and breast cancerMDA-MB-231, and one non-tumor cell line Vero, were employed incytotoxicity assay. Gelatin zymography showed that HT1080 expressed bothMMP-2 and MMP-9, A2058 only expressed MMP-2, MDA-MB-231 only expressedMMP-9, in both conditioned serum-free media and cell extracts,reflecting the gelatinases expressed by these three tumor cell lineswere secreted into the media and may also associated with the cellsurface (FIG. 2). In contrast, Vero cells had very low background of MMPexpression (FIG. 2).

Cytotoxicity of WT-PA and the PA mutants to these cells were performedonto 96-well plates. When cells grew to 80 to 100% confluence, differentconcentrations (from 0 to 1000 ng/ml) of WT-PA, PA-L1 and PA-L2 combinedwith FP59 (constant at 50 ng/ml) were separately added to the cells andchallenged the cells for 6 hours and 48 hours. For the PA dependentcytotoxicity of FP59 relies on inhibition of initial protein synthesisby ribosylating EF-2, cytotoxicity was allowed to develop for 48 hours.The EC₅₀ (concentration needed to kill half of the cells) of PA and thePA mutants were summarized in Table 1. FIG. 3a showed MMP non-expressingVero cells were quite resistant to PA-L1 and PA-L2, but very sensitiveto wild-type PA with dose-dependent manner. However, the PA-L1 and PA-L2nicked by MMP-2 in vitro efficiently killed Vero cells even with 6 hourstoxin challenge in dose-dependent manner (FIG. 3b ), demonstrating thenon toxicity of PA-L1 and PA-L2 to Vero cells was due to Vero cells lackthe ability of processing them into the active form PA63. We will showlater (in FIG. 7) that WT-PA, PA-L1 and PA-L2 quickly bound to Verocells, but only WT-PA could be processed by Vero cells to the activeform PA63, while PA-L1 and PA-L2 not. In contrast to Vero cells, the twoMMP expressing tumor cells, HT1080, A2058 and MDA-MB-231, were quitesusceptible to WT-PA as well as PA-L1 and PA-L2 (FIGS. 4a, 4b and 4c ),and the sensitivity to these PA mutants seemed directly correlated withthe overall expression levels of MMPs of these tumor cells (FIG. 2).

TABLE 1 EC₅₀ ^(a) (ng/ml) of wild type and mutated PA proteins (plus 50ng/ml FP59) on target cells Vero HT1080 A2058 MDA-MB-231 COS-7 COSgMT1WT-PA 5^(b) (6)^(c) 2.5 (5.5) 2 (6)  1 (2)   6 (15) 20 (30) PA-L1 >>1000(>>1000)  2 (10) 4 (20) 3 (15) >>1000 (>>1000) 20 (40) PA-L2 >>1000(>>1000)  2 (10) 7 (25) 4 (30) >>1000 (>>1000) 20 (20) Nicked^(d) PA-L120 Nicked^(d) PA-L2 20 ^(a)EC₅₀ is the concentration of toxin requiredto kill half of the cells compared with untreated controls. EC₅₀ valuesare interpolated from FIG. 3, 4 and 8. ^(b)EC₅₀ value for 48 hours toxintreatment ^(c)Value in parenthesis is EC50 for 6 hours toxin treatment^(d)Nicked by MMP-2

To further demonstrate the cytotoxicity of the PA mutants to the tumorcells was dependent on MMP activity expressed by the target cells, wecharacterized the effects of the well described MMP inhibitors, BB94(Batimastat), BB-2516 (Marimastat)), and GM6001, on cytotoxicity ofWT-PA, PA-L1 and PA-L2 to HT1080 cells. All these MMP inhibitors,especially GM6001, conferred clear protection to HT1080 cells againstthe challenge with PA-L1 and PA-L2 plus FP59, but did not protect thecells against WT-PA plus FP59 (FIG. 5). Thus, killing the tumor cells byPA-L1 and PA-L2 was really dependent on MMP activity expressed by thetarget cells.

PA-L1 and PA-L2 Specifically Killed MMP Expressing Tumor Cells in aCo-Culture Model

We designed a co-culture model to mimic the in vivo condition to verifywhether PA-L1 and PA-L2 specifically kill MMP expressing tumor cells,not MMP non-expressing cells. Vero, HT1080, MDA-MB-231 and A2058 cellswere cultured into the different chambers of 8-chamber slides. When thecells reached confluence, the chamber partition was removed and theslide was put into a petri culture dish with serum free medium, so thatthe different cells were in the same culture environment. PA, PA-L1 orPA-L2 (300 ng/ml) plus FP59 (50 ng/ml), or FP59 (50 ng/ml) alone wereseparately added to the cells and incubated for 48 hours forcytotoxicity assay as described in Materials and Methods. The resultshowed WT-PA unselectively killed all cells, meanwhile PA-L1 and PA-L2only killed HT1080, MDA-MB-231 and A2058 cells, but did not hurt MMPnon-expressing Vero cells (FIG. 5). This result defined the relativecontributions of membrane-associated versus soluble MMPs, indicated theactivation processing of the PA mutants mainly happened on the surfaceof the tumor cells instead of in the supernatant. Binding and processingof WT-PA, PA-L1 and PA-L2 on the surface of MMP non-expressing Verocells and MMP expressing HT1080 cells were also directly assessed. Veroand HT1080 cells were incubated with WT-PA, PA-L1 and PA-L2 for 0, 10min, 40 min, 120 min and 360 min at 37° C., respectively. Then the cellswere washed and cell lysates were prepared for western blotting analysisto check the transformation of WT-PA and PA mutants to the active formPA63. The data showed WT-PA, PA-L1 and PA-L2 could be detected in theVero and HT1080 cell lysates as soon as 10 min after incubation,demonstrating WT-PA and PA mutants could quickly bound to the cellsurface (FIG. 7a, 7b ). WT-PA was processed by both of these two celllines. In contrast, PA-L1 and PA-L2 were only processed by MMPexpressing HT1080 cells but not MMP non-expressing Vero cells (FIG. 7a,7b ), being consistent with the previous results that PA-L1 and PA-L2could only be processed by MMPs (FIGS. 1b and 1c ) and selectivelykilled MMP-expressing tumor cells (FIG. 6). Though HT1080 cellsprocessed WT-PA, PA-L1 and PA-L2, but the results showed the cellsprocessed WT-PA more efficiently than PA-L1 and PA-L2 (FIG. 7b ),reflecting the activity of furin or furin-like proteases was higher thanthat of MMPs on the cell surface. We also analyzed the processing statusof PA-L1 and PA-L2 in the culture supernatants of HT1080 cells, andcould not detect their active form PA63 in the overnight culturesupernatants, but with time increasing the randomly breakdown productsshowed up (data not shown).

MT1-MMP Played a Role in Activation of PA-L1 and PA-L2

Zymographic analysis showed COS-7 cells expressed very negligible amountgelatinases (FIG. 8a insert). Thus, just as expected, COS-7 cells wereresistant to PA-L1 and PA-L2 plus FP59, but susceptible to WT-PA plusFP59 (FIG. 8a ). To examine the role of MT1-MMP in activation of PA-L1and PA-L2, encoding sequence of MT1-MMP was transfected into COS-7cells, resulting in a stable transfectant COSgMT1 in which expression ofMT1-MMP was detected by western blotting (FIG. 8b insert). In contrastto COS-7 cells, COSgMT1 became very sensitive to PA-L1 and PA-L2 (FIG.8b ), indicating MT1-MMP played a role in activation of these PAmutants, either by directly processing the cell bound PA mutants, or byindirect way that activated pro-MMP-2 or other MMPs first, which in turnprocessed PA mutants to their active form PA₆₃. It seemed unlikely thelater one, for COS-7 cells expressed negligible amount of MMPs.

Example II Construction of Mutant PA with Matrix MetalloproteinaseCleavage Sites

Mutant PA proteins were constructed and tested as described in ExampleI, substituting one of the following plasminogen activator cleavagesites of Table 2 for the MMP cleavage sites described above. Phagedisplay libraries were used to identify sequences having specificity fora particular protease (see, e.g., Coombs et al., J. Biol. Chem.273:4323-4328 (1998); Ke et al., J. Biol. Chem. 272:20456-20462 (1997);Ke et al., J. Biol. Chem. 272:16603-16609 (1997)). These libraries canbe used by one of skill in the art to select sequences specificallyrecognized by MPP and plasminogen activator proteases.

TABLE 2 u-TP and t-PA cleavage sites Substrate SEQ ID u-PA t-PAa-PA:t-PA sequence NO: Kcat/Km Kcat/Km selectivity PCPGRVVGG 4 0.88 0.293.0 PGSGRSA 5 1200 60 20 PGSGKSA 6 193 1.6 121 PQRGRSA 7 45 850 0.005

Example III Construction of Mutant PA with Plasminogen ActivatorCleavage Sites

A. Materials

Enzymes for DNA manipulation and modification were purchased from NewEngland Biolabs (Beverly, Mass.). FP59 and a soluble form of furin wereprepared in our laboratory as described (Gordon, V. M., et al., Infect.Immun., 65:4130-4134 (1997)). Rabbit anti-PA polyclonal antibody (#5308)was made in our laboratory. Pro-uPA (single-chain uPA, #107), uPA(#124), tPA (#116), human urokinase amino-terminal fragment (ATF)(#146), human glu-plasminogen (#410), human PAI-1 (#1094), human plasmin(#421), monoclonal antibody against human uPA B-chain (#394) werepurchased from America Diagnostica inc (Greenwich, Conn.). Goatpolyclonal antibody against human t-PA (sc-5241) was purchased fromSanta Cruz Biotechnology, Inc. (Santa Cruz, Calif.). uPAR monoclonalantibody R3 was a gift

Construction of Mutated PA Proteins

A modified overlap PCR method was used to construct the mutated PAproteins in which the furin site is replaced by the uPA and tPAphysiological substrate sequence PCPGRVVGG (SEQ ID NO:4) in PA-U1, uPAfavorite sequences PGSGRSA (SEQ ID NO:5) and PGSGKSA (SEQ ID NO:6) inPA-U2 and PA-U3, respectively, tPA favorite sequence PQRGRSA (SEQ IDNO:7) in PA-U4. The PA expression plasmid pYS5 (Singh, Y., et al., JBiol Chem, 264:19103-19107 (1989)) was used as template. A 5′ primer F,AAAGGAGAACGTATATGA (SEQ ID NO:8) (Shine-Dalgarno and start codons areunderlined), and the phosphorylated reverse primer R1, pTGGTGAGTTCGAAGATTTTTGTTTTAATTCTGG (SEQ ID NO:13) (the first three nucleotides encodeP, the others anneal to the sequence corresponding to P₁₅₄-S₁₆₃), wereused to amplify a fragment designated “N”. A mutagenic phosphorylatedprimer H1, pTGTCCAGGAAG AGTAGTTGGAGGAAGTACAAGTGCTGGACCTACGGTTCCAG (SEQID NO:14), encoding CPGRVVGG (SEQ ID NO:15) and S₁₆₈-P₁₇₆, and reverseprimer R2, ACGTTTATCTCTTATTAAAAT (SEQ ID NO:11), annealing to thesequence encoding I₅₈₉-R₅₉₅, were used to amplify a mutagenic fragment“M1”. A phosphorylated mutagenic primer H2,pGGAAGTGGAAGATCAGCAAGTACAAGTGCTGGACCTACGGTTCCAG (SEQ ID NO:16), encodingGSGRSA (SEQ ID NO:17) and S₁₆₈-P₁₇₆, and reverse primer R2 were used toamplify a mutagenic fragment “M2”. A phosphorylated mutagenic primer H3,pGGAAGTGGAAAATCAGCAAGTACAAGTGCTGGACCTACGGTTCCAG (SEQ ID NO:18), encodingGSGKSA (SEQ ID NO:19) and S₁₆₈-P₁₇₆, and reverse primer R2, were used toamplify a mutagenic fragment “M3”. A phosphorylated mutagenic primer H4,pCAGAGAGGAAGATCAGCAAGTACAAGTG CTGGACCTACGGTTCCAG (SEQ ID NO:20),encoding QRGRSA (SEQ ID NO:21) and S₁₆₈-P₁₇₆, and reverse primer R2,were used to amplify a mutagenic fragment “M4”. Primers F and R2 wereused to amplify the ligated products of N+M1, N+M2, N+M3, and N+M4,respectively, resulting in the mutagenized fragments U1, U2, U3, and U4in which the coding sequence for the furin site (RKKR₁₆₇; SEQ ID NO:1)is replaced by uPA or tPA substrate. The HindIII/PstI digests of U1, U2,U3, and U4 were cloned between the HindIII and PstI sites of pYS5. Theresulting expression plasmids were named pYS-PA-U1, pYS-PA-U2,pYS-PA-U3, and pYS-PA-U4, and their expression products, the mutated PAproteins, were accordingly named PA-U1, PA-U2, PA-U3, and PA-U4. Oneexpression plasmid encoded a mutant in which RKKR₁₆₇ (SEQ ID NO:1) isreplaced by PGG, expected not to be cleaved by any protease. Itsexpression plasmid and expression product were named pYS-PA-U7 andPA-U7, respectively.

Expression and Purification of PA and Mutated PA Proteins

To express PA, PA-U1, PA-U2, PA-U3, PA-U4, and PA-U7, the expressionplasmids pYS5, pYS-PA-U1, pYS-PA-U2, pYS-PA-U3, pYS-PA-U4, andpYS-PA-U7, were transformed into non-virulent strain B. anthracisUM23C1-1 and grown in FA medium (Singh, Y., et al., J. Biol. Chem.,264:19103-19107 (1989)) with 20 μg/ml of kanamycin for 16 h at 37° C.The expression products were secreted into the culture supernatants. Themutated PA proteins were concentrated and purified by chromatography ona MonoQ column (Amersham Pharmacia Biotech, Piscataway, N.J.), asdescribed previously (Varughese, M., et al., Mol. Med., 4:87-95 (1998)).

In Vitro Cleavage of PA and Mutated PA Proteins by uPA, tPA, and Furin

Reaction mixtures of 50 μl containing 5 μg of the PA proteins wereincubated at 37° C. with 5 μl of soluble furin or 0.5 μg of uPA or tPA.Furin cleavage was done in 25 mM HEPES, pH 7.4, 150 mM NaCl, 0.2 mMEDTA, 0.2 mM EGTA, 100 μg/ml ovalbumin, 1.0 min CaCl₂, and 1.0 mM MgCl₂.Aliquots (5 μl) withdrawn at intervals were separated by polyacrylamidegel electrophoresis (PAGE) using 10-20% gradient Tris-glycine gel(Novex, San Diego, Calif.) and visualized by Commassie staining.Cleavage with uPA or tPA was done in 150 mM NaCl, 10 mM Tris-HCl (pH7.5). Aliquots withdrawn at intervals were diluted 1:1000 and separatedby PAGE using 10-20% gradient Tris-glycine gel (Novex, San Diego,Calif.) and electroblotted to a nitrocellulose membrane (Novex, SanDiego, Calif.). Cleavage was assessed by Western blotting with a rabbitanti-PA antibody. Membranes were blocked with 5% (w/v) non-fat milk,incubated sequentially with rabbit anti-PA polyclonal antibody (#5308)and horse radish peroxidase-conjugated goat anti-rabbit antibody(sc-2004, Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.), andvisualized by ECL (Pierce, Rockford, Ill.).

Cells and Culture Medium

Vero cells, human cervix adenocarcinoma Hela cells, human melanoma A2058cells, human melanoma Bowes cells, and human fibrosarcoma HT1080 cellswere obtained from American Type Culture Collection (Manassas, Va.). Allcells were grown in Dulbecco's Minimal Essential Medium (DMEM) with0.45% glucose, 10% fetal bovine serum, 2 mM glutamine, and 50 μg/mlgentamicin. Human primary vascular endothelial cells were obtained andcultured according to standard methodology. Cells were maintained at 37°C. in a 5% CO₂ environment.

Binding and Processing of Pro-PA by Cultured Cells

Vero cells, Hela cells, A2058 cells, and Bowes cells were cultured in24-well plate to confluence, washed and incubated in serum-free mediawith 1 μg/ml of pro-uPA and 1 μg/ml of glu-plasminogen for 1 h, then thecell lysates were prepared for Western blotting analysis with monoclonalantibody against uPA B-cahin (#394).

Cytotoxicity Assay with MTT

Cells were seeded into 96-well plates at approximately 25% confluence.The next day, cells were washed twice with serum-free DMEM to removeresidual serum. Serial dilutions of PA, mutated PA proteins (0 to 1000ng/ml) combined with FP59 (50 ng/ml) in serum-free DMEM (If targetingurokinase plasminogen activation system, 100 ng/ml pro-uPA and 1 μg/mlof glu-plasminogen were added) to the cells to give a total volume of200 μl/well. In some experiments, PM-1 was added 30 min prior to toxinaddition. Cells was incubated with the toxins for 6 h, after which themedium was replaced with fresh DMEM supplemented with 10% FCS. Cellviability was then assayed by adding 50 μl of 2.5 mg/ml MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide). Thecells were incubated with MTT for 45 min at 37° C., the medium wasremoved, and the blue pigment produced by viable cells was solubilizedwith 100 μl/well of 0.5% (w/v) SDS, 25 mM HCl, in 90% (v/v) isopropanol.The plates were vortexed and the oxidized MTT was measured as A₅₇₀ usinga microplate reader.

Binding and Processing of PA and PA-U2 by Cultured Cells

Cells were grown in 24-well plates confluence and washed twice withserum-free DMEM to remove residual serum. Then the cells were incubatedwith 1 μg/ml of PA and PA-U2 at 37° C. in serum-free DMEM containing 100ng/ml of pro-uPA and 1 μg/ml of glu-plasminogen for different lengths oftime. When PAI-1 was tested, it was incubated with cells for 30 minprior to the addition of PA proteins. The cells were washed five timesto remove unbound PA proteins. Cells were lysed in 100 μl/well modifiedRIPA lysis buffer (50 mM Tris-HCl, pH 7.4, 1% NP40, 0.25%Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethyl sulfonylfluoride, 1 μg/ml each of aprotinin, leupeptin and pepstatin) on ice for10 min. Equal amounts of protein from cell lysates were separated byPAGE using 10-20% gradient Tris-glycine gels (Novex, San Diego, Calif.).Western blotting to detect PA and its cleavage products was performed asdescribed above.

Cytotoxicity Assay in a Co-Culture System

A co-culture model was designed to mimic the in vivo condition to verifywhether PA-U2 kill uPAR-overexpressing tumor cells while not affectinguPAR non-expressing cells. Vero, Hela cells were cultured in separatechambers of 8-chamber slides (Nalge Nunc International, Naperville,Ill.) to 80-100% confluence. The cells were washed twice with serum-freeDMEM, the chamber partition was removed, and the slide was put into aculture dish with serum-free medium containing 100 ng/ml pro-uPA and 1μg/ml of Glu-plasminogen, so that all the cells were bathed in the samemedium. PA and PA-U2 (300 ng/ml) and FP59 (50 ng/ml) were addedindividually or in combination and cells were exposed for 48 h. Then MTT(0.5 mg/ml) was added for 45 min at 37° C., the partitions wereremounted, and the oxidized MTT in each chamber was dissolved asdescribed above to determine the viability of each cell type. The celllysates from different chambers were also prepared for Western blottingto detect PA proteins and their cleavage product PA63 species.

B. Results

Directing uPA or tPA Sequence-Specific Proteolysis to Anthrax PA

The crystal structure of PA shows that the furin site, RKKR₁₆₇ (SEQ IDNO:1), is in a surface-exposed, flexible loop composed of aa 162 to 175(Petosa, C., et al., Nature, 385:833-838 (1997)). Cleavage in this loopby furin or furin-like proteases is essential to toxicity. Mutated PAproteins were constructed in which the furin-sensitive sequence RKKR₁₆₇(SEQ ID NO:1) is replaced by uPA or tPA substrate sequences. In mutatedPA protein PA-U1, PCPGRVVGG (SEQ ID NO:4), a peptide from P5 to P4′ inthe physiological substrate plasminogen, was used to replace RKKR₁₆₇(SEQ ID NO:1). In PA-U2, RKKR₁₆₇ (SEQ ID NO:1) was replaced by apeptide, PGSGRSA (SEQ ID NO:5), containing the consensus sequence SGRSA(SEQ ID NO:22) from P3 to P2′, which was recently identified as theminimized best substrate for uPA (Ke, S. H., et al., J. Biol. Chem.,272:20456-20462 (1997)). Because the peptide SGRSA (SEQ ID NO:22) iscleaved 1363-fold times more efficiently than a control peptidecontaining the physiological cleavage site present in plasminogen byuPA, and exhibits a uPA/tPA selectivity of 20 (Ke, S. H., et al., J.Biol. Chem., 272:20456-20462 (1997)), PA-U2 was expected to be afavorite substrate of uPA. uPA/tPA selectivity of the peptide SGRSA (SEQID NO:22) can be further enhanced by placement of lysine in the P1position (Ke, S. H., et al., J. Biol. Chem., 272:20456-20462 (1997)),thus, the peptide PGSGKSA (SEQ ID NO:6), which exhibits a uPA/tPAselectivity of 121 (Ke, S. H., et al., J. Biol. Chem., 272:20456-20462(1997)), was used to replace RKKR₁₆₇ (SEQ ID NO:1) to construct amutated PA protein, PA-U3, with even higher uPA selective activity thanPA-U2. The investigation showed P3 and P4 residues were the primarydeterminants of the ability of a substrate to discriminate between tPAand uPA, and mutation of both P4 glycine and P3 serine of the mostlabile uPA substrate (GSGRSA; SEQ ID NO:17) to glutamine and arginine,respectively, decreased the uPA/tPA selectivity by a factor of 1200 andactually converted the peptide into a tPA-selective substrate (Ke, S.H., et al., J. Biol. Chem., 272:20456-20462 (1997)). Based on thisstudy, a mutated PA protein, PA-U4, was constructed. PA-U4 is expectedto be a tPA favorite substrate, in which the peptide PQRGRSA (SEQ IDNO:7) was used to replace RKKR₁₆₇ (SEQ ID NO:1). A mutated PA proteinPA-U7, was also constructed in which RKKR₁₆₇ (SEQ ID NO:1) was replacedby random sequence PGG, expected not to be cleaved by any knownproteases, was used a control protein in this study. The designations ofthe mutated PA proteins along with the expected properties weresummarized in Table 3.

Plasmids encoding these mutated PA proteins were constructed by amodified overlap PCR method, cloned into the E. coli-Bacillus shuttlevector pYS5, and efficiently expressed in B. anthracis UM23C1-1. Theexpression products were secreted into the culture supernatants at 20-50mg/L. The mutated PA proteins were concentrated and purified by MonoQchromatography to one prominent band at the expected molecular mass of83 kDa which co-migrated with PA in SDS-PAGE. Thus, using a productionprotocol that is now standard for PA, these mutated PA proteins could beexpressed and purified easily, in high yield and purity.

To verify that the mutated PA proteins had the expected susceptibilityto proteases, they were subjected to cleavage with a soluble form offurin, uPA and tPA. As expected, these mutated PA proteins, hadcompletely lost the susceptibility to furin. In contrast, wild-type PAwas very sensitive to furin and processed to the active form PA63 (FIG.9a ). The cleavage profiles of these mutated PA proteins by uPA and tPAwere quite consistent with that obtained from the peptide substrates(FIG. 9b, 9c ). PA-U2 was efficiently cleaved by uPA, which was followedby PA-U3. PA-U3 could only be cleaved by uPA, but not tPA, showing highuPA specificity. However, PA-U2 was also slightly cleaved by tPA, beinga week substrate for tPA. In contrast, PA-U4 was a very week substratefor uPA, but a good substrate for tPA. PA-U7 as well as PA-U1 were bothcompletely resistant to uPA and tPA. PA was completely resistant to tPA,but was a week substrate for uPA (FIG. 9b ). These results implicatedPA-U2 and PA-U3 which can be selectively activated by uPA may be usefulto target tumor cell surface-associated plasminogen activation systemfor tumor therapy, while PA-U4 may be toxic to tPA expressing cellswhich usually occurred in neuroblastomas.

PA-U2 and PA-U3 Selectively Kill Tumor Cells by Targeting Tumor CellSurface-Associated Plasminogen Activation System

uPAR is typically overexpressed in tumor cell lines and tumor tissues,and is the central part of cell surface-associated plasminogenactivation system which is essential to tumor invasion and metastasis.To test the hypothesis that PA-U2 and PA-U3 would preferentially killuPAR-overexpressing tumor cells, cytotoxicity assays were performed withthree human tumor cell lines: cervix adenocarcinoma Hela, melanomaA2058, and melanoma Bowes. A non-tumor monkey cell line, Vero, was usedas control. The expression of uPAR by these three tumor cell lines butnot by Vero cells was evidenced by binding and processing of pro-uPA tothe active form two-chain uPA by these three tumor cells but not by Verocells. FIG. 10 showed that after 1 h incubation with the cells, pro-uPAand the processed form uPA B-chain could be detected from these threetumor cell lysates but not from Vero cells.

Cytotoxicity of PA and the mutated PA proteins to these cells wasmeasured in 96-well plates. In tumor tissues, tumor cells typicallyoverexpress uPAR, while tumor stromal cells express pro-uPA which bindsand thereby is activated on the tumor cell surface, therefore in thecytotoxicity assay 100 ng/ml of pro-uPA was added to the tumor cells tomimic the role of tumor stromal cells in vivo. In addition, plasminogenis an important component of plasminogen activation system, and presentat high concentration (1.5-2.0 μM) in plasma and interstitial fluids,representing potential plentiful source of plasmin activity. Therefore.1 μg/ml of glu-plasminogen was also added in the cytotoxicity assay. PAand the mutated PA proteins combined with FP59 were incubated with cellsfor 6 h, and the viability was measured after 48 h. The EC₅₀ values(concentrations needed to kill half of the cells) for PA and the mutatedPA proteins are summarized in Table 4. The three uPAR-expressing tumorcells, Hela, A2058, and Bowes were very susceptible to PA as well as toPA-U2 and PA-U3, and less susceptible to PA-U4 (FIG. 11 a, b, c). Incontrast, these tumor cells were completely resistant to PA-U1 and PA-U7(FIG. 11 a, b, c). The order of the cytotoxicity of mutated PA proteinsto these tumor cells: PA-U2>PA-U3>PA-U4>>PA-U1, PA-U7, was wellcorrelated with the uPA cleavage profile showed in FIG. 9b . In contrastto the tumor cells, the uPAR non-expressing Vero cells were completelyresistant to all the mutated PA proteins, but sensitive to PA in adose-dependent manner (FIG. 12a ). However, PA-U2 that was first nickedby uPA in vitro efficiently killed Vero cells (FIG. 11b ). Thisdemonstrated that the resistance of Vero cells to PA-U2 was due to theinability of the cells to proteolytically activate the mutated PAproteins.

Binding and proteolytically processing of PA and PA-U2 on cell surfacewere also assessed. Vero and Hela cells were incubated with PA and PA-U2for various length of times. After that the cell lysates were preparedand examined by Western blotting to detect binding and processing statusof the PA proteins to the active PA63 species. PA was processed by bothcell types, and this could not be inhibited by PAI-1 (FIG. 13a, b ). Incontrast, PA-U2 was processed by Hela cells but not by Vero cells, andthis could be completely blocked by PAI-1 (FIG. 13a, b ), demonstratingthe cleavage of PA-U2 on Hela cell surface was due to uPA activated onthe surface. Although Hela cells proteolytically processed PA as well asPA-U2, the later was cleaved slower apparently due to its cleavage wassecondary to pro-uPA activation (FIG. 13b ).

To further demonstrate that the cytotoxicity of the mutated PA proteinsfor tumor cells was dependent on the tumor cell surface-associatedplasminogen activation system, the effects of the specific inhibitor andblockers of the system were characterized. PAI-1 conferred strongprotections to all these three tumor cells against challenge with PA-U2plus FP59, but did not protect the cells from PA plus FP59 (FIG. 14 a,b, c). ATF, the amino-terminal fragment and uPAR binding domain of uPA,which competes the binding site on uPAR with pro-uPA, protected allthree tumor cells from PA-U2 plus FP59 with dose-dependent manner (FIG.15a ). Similarly, uPAR blocking monoclonal antibody R3 whichspecifically interferes the binding between pro-uPA and uPAR, alsoprotected the tumor cells in all three cases from PA-U2 plus FP59 (FIG.15b ). These results demonstrated killing of these tumor cells by PA-U2was dependent on tumor cell surface-associated plasminogen activationsystem.

PA-U2 Retained Selectivity for uPAR-Expressing Cells in a Co-CultureModel

A co-culture model was designed to mimic in vivo conditions, to testwhether PA-U2 can selectively kill Hela cells but not the bystandercells. Vero and Hela cells were cultured in separate compartments of8-chamber slides. When the cells reached confluence, the chamberpartitions were removed and the slides were put into culture dishes withserum-free medium containing 100 ng/ml of pro-uPA and 1 μg/ml ofglu-plasminogen so that all cells on the slide were bathed in the samemedium. PA and PA-U2 (each at 300 ng/ml) plus FP59 (50 ng/ml), or FP59alone were added to the culture dishes and incubated for 48 h beforemeasuring viability. The results showed that PA was processed to activePA63 by and killed both cells, whereas PA-U2 was processed to activePA63 by and killed only Hela cells, while not affecting the uPARnon-expressing Vero cells (FIG. 16. inset). These results showed thatPA-U2 is not activated in the tissue culture medium by uPAR unbound uPA,nor do PA proteins proteolytically activated on the surface of one celldissociate and rebind on other cells. Activate uPA in the culturesupernatant would have led to killing of the Vero cells, because FIG.12b showed that PA-U cleaved in solution became cytotoxic.

PA-U4 was Toxic to tPA Expressing Cells while PA-U2 and PA-U3 are not

FIG. 9 showed PA-U4 is a good substrate of tPA among these mutated PAproteins and expected to be toxic to tPA expressing cells. To test thishypothesis, cytotoxicity assay was performed on two tPA expressingcells: human melanoma Bowes, and human primary vascular endothelialcells (HUVEC). The expression of tPA by these cells was evidenced byWestern blotting analysis of the culture supernatants by using apolyclonal antibody against human tPA (data not shown). The cells werecultured to 50% confluence, then cytotoxicity assay were done inserum-free DMEM not containing pro-uPA and glu-plasminogen. Differentconcentrations (from 0 to 1000 ng/ml) of PA, PA-U2, PA-U3, and PA-U4combined with FP59 (50 ng/ml) were incubated with cells for 12 h, andviability was measured after 48 h. The EC₅₀ values for the PA proteinswere summarized in Table 5. PA-U4 was toxic to the two tPA expressingcells, while PA-U2 and PA-U3 showed a very low toxicity to them (FIG.17a, b and Table 5). These and the above results clearly showed that uPAand tPA susceptibility differentiate among these mutated PA proteins.PA-U2 and PA-U3 which specifically target tumor cell surface-associatedplasminogen activation system may be very useful for tumor therapy.While PA-U4 which could be activated by tPA may be applied for someneurosystem tumors which usually overexpress tPA.

Discussion

Increasing evidence has been accumulated that the components of theurokinase plasminogen activation system are involved in tumor cellproliferation, invasion, and metastasis since 1976 when it wasdiscovered that uPA was produced and released from cancer cells(Schmitt, M., et al., Thromb. Haemost., 78:285-296 (1997)). Recent datasuggested that invasion factors may also serve as targets for newtreatments to prevent cancer invasion and metastasis (Schmitt, M., etal., Thromb. Haemost., 78:285-296 (1997)). Various different approachesto interfere with the expression or the activity of uPA, uPAR, and PAI-1at gene or protein level were successfully tested in vitro or in miceincluding antisense oligonucleotides, antibodies, inhibitors, andrecombinant or synthetic uPA and uPAR analogues (Schmitt, M., et al.,Thromb. Haemost, 78:285-296 (1997)). However, it is expected that theseapproaches should only slow the growth of tumors, without having adirect cytotoxic action that could eradicate the malignant cells. Thepresent study is the first to exploit the tumor cell surface associatedplasminogen system to achieve cell-type selective targeting of cytotoxicbacterial toxin fusion proteins. In this study, mutated anthrax toxinprotective antigen (PA) proteins, PA-U2, PA-U3, and PA-U4, wereconstructed in which the furin recognition site is replaced bysusceptible sequences cleaved by uPA (PA-U2 and PA-U3) or tPA (PA-U4)more efficiently than control peptides containing the physiologicaltarget sequence present in plasminogen. More interestingly is that thesusceptibility toward uPA and tPA differentiated among these mutated PAproteins, i.e., PA-U2 and PA-U3 were mainly activated by uPA, whilePA-U4 was mainly activated by tPA. Thus, when combined with FP59, arecombinant fusion toxin derived from anthrax lethal factor andPseudomonas exotoxin A, PA-U2 and PA-U3 selectively killeduPAR-overexpressing tumor cells in the present of pro-uPA, and meanwhileshowed very low toxicity to tPA expressing cells such as vascularendothelial cells. Because tPA is secreted as an active enzyme mainly byvascular endothelial cells in vivo (Mann, K., et al., Annu. Rev.Biochem., 57:915-956 (1988)), the cytotoxicity differentiation amongthese mutated PA proteins to uPA and tPA expression cells is soimportant to avoid the damage to the vascular endothelial cells whenPA-U2 and PA-U3 are used in vivo.

The following lines of evidence clearly demonstrate that the proteolyticactivation of these uPA-activated mutated PA proteins occurred on thetumor cell surface that was dependent upon the activity of tumor cellsurface associated plasminogen activation system: 1. Pro-uPA could onlybind and thereby proteolytically activated on uPAR-expressing tumor cellsurface but not on uPAR non-expressing Vero cells; 2. PA-U2 could onlybe proteolytically processed to the active form PA63 on uPAR-expressingcells (such as Hela cells) but not on uPAR non-expressing Vero cells,and this processing could be completely inhibited by uPA specificinhibitor PAI-1; 3. The toxicity of PA-U2 to the tumor cells waseliminated by uPAR specific blocking reagent ATF, uPAR blocking antibodyR3, and PAI-1, demonstrating the activation of PA-U2 was entirelydependent upon the activation of pro-uPA on tumor cell surface; 4.Cytotoxicity assays in a co-culture model, in which the cells wereequally accessible to the toxins in the supernatant, showed that PA-U2killed only uPAR-overexpressing Hela cells and not the bystander Verocells, demonstrating that activation of uPA-activated mutated PAproteins occurred principally on cell surfaces, because the active formof PA proteins in solution could also kill the Vero cells.

PA proteins bind to cells rapidly and with high affinity (Kd approx. 1nM), therefore, even at low PA concentrations, PA receptors will behighly occupied. As a result, if there were any PA which becameactivated in the supernatant or dissociated from a cell after cleavagewould be unable to locate a free receptor by which to bind to cells andinternalize FP59.

Thus, the cytotoxicity of these cytotoxins was directed selectively tothe uPAR-overexpressing tumor cells. PA-U4, which could be activated bytPA, can be applied for intratumoral therapy of some unresectableneurosystem tumors which usually overexpress tPA.

Tumor-cell selective cytotoxins have been created by replacing thereceptor-recognition domains of bacterial and plant protein toxins withcytokines, growth factors, and antibodies (Kreitman, R. J., Curr. Opin.Immunol., 11:570-578 (1999)). The protein toxins used contain anenzymatic domain that acts in the cytosol to inhibit protein synthesisand a domain which achieves translocation of this catalyst from avesicular compartment to the cytosol, as well as the cell-targetingdomain that is replaced or altered so as to achieve tumor cellspecificity. Certain of these “immunotoxins” derived from diphtheriatoxin, Pseudomonas exotoxin A, and ricin have shown efficacy and havebeen approved for clinical use. However, a recurrent problem with thesematerials is that therapeutic doses typically damage other tissues andcells (Frankel, A. E., et al., Semin. Cancer Biol., 6:307-317 (1995)).This is not surprising because very few of the tumor cell surfacereceptors or antigens that are targeted are totally absent from normaltissue. Therefore, even in the best cases, some toxin uptake will occurin normal bystander cells. Because these toxins act catalytically, evena small amount of internalized toxin can seriously damage normal tissue.Even a single molecule delivered to the cytosol can kill a cell(Yamaizumi, M., et al., Cell, 15:245-250 (1978)). Previous efforts todevelop anthrax toxin fusion proteins as therapeutic agents have focusedon modification of domain 4, the receptor-binding domain of PA. Work isongoing to create cell-type specific cytotoxic agents by modifying orreplacing domain 4 to direct PA to alternate receptors (Varughese, M.,et al., Mol. Med., 4:87-95 (1998); Varughese, M., et al., Infect.Immun., 67:1860-1865 (1999). This work follows the example of thedevelopment of immunotoxins from other protein toxins, as cited earlier(Kreitman, R. J., Curr. Opin. Immunol., 11:570-578 (1999)). We suggestthat combining two conceptually distinct targeting strategies in asingle PA protein will yield agents having higher therapeutic indices. Aprotein that is both retargeted to a tumor cell surface protein anddependent on cell surface plasminogen activation system for activationmay achieve therapeutic effects while being free of the side effectsobserved with many of the existing immunotoxins.

TABLE 3 PA proteins generated in this study Desig- SEQ ID K_(cat)/Km¹uPA:tPA Protease expected nation Sequence at the ″furin loop″ NO: uPAtPA selectivity¹  to cleave PA NS RKKR↑ STSAGPTV 23 Furin PA-U1NSPCPGR↑ VVGG STSAGPTV 24 0.88 0.29 3 uPA/tPA  (weakly) PA-U2NSPGSGR↑ SA STSAGPTV 25 1200 60 20 uPA PA-U3 NSPGSGK↑ SA STSAGPTV 26 1931.6 121 uPA PA-U4 NSPQRGR↑ SA STSAGPTV 27 7.3 670 0.005 tPA PA-U7 NSPGGSTSAGPTV 28 None ¹Data was cited from Ke, S. H., et al., J. Biol. Chem.,272:20456-20462 (1997) which was obtained from the studies on thepeptides underlined in column 2.

TABLE 4 Toxicities (EC₅₀ in μg/ml) of PA proteins to various cells Cellline Cell type PA PA-U2 PA-U3 PA-U4 Hela Human cervix 12 14 30 200adenocarcinoma cell line A2058 Human melanoma cell line 10 13 18 50Bowes Human melanoma cell line 7 8 15 50 Vero Monkey kidney normal15 >1000 >1000 >1000 epithelial cell lineEC₅₀ is the concentration of toxin required to kill half of the cells.EC₅₀ values are interpolated from FIGS. 11 and 12.

TABLE 5 Toxicities (EC₅₀ in ng/ml) of PA proteins to tPA expressingcells Cell line Cell type PA PA-U2 PA-U3 PA-U4 HUVEC Human primaryvascular <1 >1000 >1000 25 endothelial cells Bowes Human melanoma cellline 3 600 >1000 12EC₅₀ is the concentration of toxin required to kill half of the cells.EC₅₀ values are interpolated from FIG. 17.

What is claimed is:
 1. An isolated mutant protective antigen proteincomprising a matrix metalloproteinase-recognized cleavage site in placeof the native protective antigen furin-recognized cleavage site, whereinthe mutant protective antigen is cleaved by a matrix metalloproteinaseselected from the group consisting of MMP-2 (gelatinase A), MMP-9(gelatinase B), and MT1-MMP (membrane-type 1 MMP), wherein the matrixmetalloproteinase-recognized cleavage site is GPLGMLSQ (SEQ ID NO:2) orGPLGLWAQ (SEQ ID NO:3).
 2. A composition comprising the mutantprotective antigen protein of claim 1 and a matrix metalloproteinaseselected from the group consisting of MMP-2, MMP-9, and MT1-MMP.
 3. Acomposition comprising the mutant protective antigen protein of claim 1and a cell expressing on its surface a matrix metalloproteinase selectedfrom the group consisting of MMP-2, MMP-9, and MT1-MMP.
 4. Thecomposition of claim 3, wherein the cell is a cell derived from human.5. The composition of claim 3, wherein the cell is a cancer cell.